Multiplexed molecular analysis apparatus and method

ABSTRACT

A method and apparatus for analyzing molecular structures within a sample substance using an array having a plurality of test sites upon which the sample substance is applied. The invention is also directed to a method and apparatus for constructing molecular arrays having a plurality of test sites. The invention allows for definitive high throughput analysis of multiple analytes in complex mixtures of sample substances. A combinatorial analysis process is described that results in the creation of an array of integrated chemical devices. These devices operate in parallel, each unit providing specific sets of data that, when taken as a whole, give a complete answer for a defined experiment. This approach is uniquely capable of rapidly providing a high density of information from limited amounts of sample in a cost-effective manner.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made at least in part with funds from the NationalAeronautics and Space Administration, Grant Number NAGW 4530.

This application is based on US provisional application 60/034,627,filed Dec. 31, 1996, incorporated herein by reference.

TECHNICAL FIELD

This invention relates to a multiplexed molecular analysis apparatus andmethod for the detection and quantification of one or more molecularstructures in a sample.

BACKGROUND

It is very desirable to rapidly detect and quantify one or moremolecular structures in a sample. The molecular structures typicallycomprise ligands, such as antibodies and anti-antibodies. Ligands aremolecules which are recognized by a particular receptor. Ligands mayinclude, without limitation, agonists and antagonists for cell membranereceptors, toxins, venoms, oligosaccharides, proteins, bacteria andmonoclonal antibodies. For example, DNA or RNA sequence analysis is veryuseful in genetic and infectious disease diagnosis, toxicology testing,genetic research, agriculture and pharmaceutical development. Likewise,cell and antibody detection is important in numerous diseasediagnostics.

In particular, nucleic acid-based analyses often require sequenceidentification and/or analysis such as in vitro diagnostic assays andmethods development, high throughput screening of natural products forbiological activity, and rapid screening of perishable items such asdonated blood, tissues, or food products for a wide array of pathogens.In all of these cases there are fundamental constraints to the analysis,e.g., limited sample, time, or often both.

In these fields of use, a balance must be achieved between accuracy,speed, and sensitivity in the context of the constraints mentionedearlier. Most existing methodologies are generally not multiplexed. Thatis, optimization of analysis conditions and interpretation of resultsare performed in simplified single determination assays. However, thiscan be problematic if a definitive diagnosis is required since nucleicacid hybridization techniques require prior knowledge of the pathogen tobe screened. If symptoms are ambiguous, or indicative of any number ofdifferent disease organisms, an assay that would screen for numerouspossible causative agents would be highly desirable. Moreover, ifsymptoms are complex, possibly caused by multiple pathogens, an assaythat functioned as a "decision tree" which indicated with increasingspecificity the organism involved, would be of high diagnostic value.

Multiplexing requires additional controls to maintain accuracy. Falsepositive or negative results due to contamination, degradation ofsample, presence of inhibitors or cross reactants, and inter/intrastrand interactions should be considered when designing the analysisconditions.

Conventional Technologies and Limitations

Sanger Sequencing

Of all the existing techniques, one of the most definitive is thetraditional Sanger sequencing technique. This technique is invaluablefor identifying previously unknown or unsuspected pathogens. It is alsovaluable in determining mutations that confer drug resistance tospecific strains of disease organisms. These analyses are generallyresearch oriented. The end result of this research, e.g., sequencedetermination of a specific pathogen, can be used to design probes foridentification applications in a clinical setting.

However, there are constraints to employing this technique in a clinicallab. The primary constraints are cost and throughput due to the inherentlabor intensive procedures, requiring multiple steps to be performed byskilled personnel. For example, typical analysis usually requires morethan a day for completion. Of more concern is the potential forambiguity when multiple strains of a pathogen are present in one sample.Virulence of the pathogen is often determined by the strain. An exampleis HPV, also known as human papilloma virus. Seventy strains of HPV arecommonly known to exist. Two strains, in particular, are stronglyassociated with an increased risk of cervical cancer, hence theaggressiveness of treatment or screening for malignancy is determined bythe presence of an HPV strain. Multiple strains cause indeterminateresults when using sequencing methodologies. The ideal assay would bemultiplexed with the selectivity to identify all strains involved.

Blotting Techniques

Blotting techniques, such as those used in Southern and Northernanalyses, have been used extensively as the primary method of detectionfor clinically relevant nucleic acids. The samples are prepared quicklyto protect them from endogenous nucleases and then subjected to arestriction enzyme digest or polymerase chain reaction (PCR) analysis toobtain nucleic acid fragments suitable for the assay. Separation by sizeis carried out using gel electrophoresis. The denatured fragments arethen made available for hybridization to labeled probes by blotting ontoa membrane that binds the target nucleic acid. To identify multiplefragments, probes are applied sequentially with appropriate washing andhybridization steps. This can lead to a loss of signal and an increasein background due to non-specific binding. While blotting techniques aresensitive and inexpensive, they are labor intensive and dependent on theskill of the technician. They also do not allow for a high degree ofmultiplexing due to the problems associated with sequential applicationsof different probes.

Microplate Assays

Microplate assays have been developed to exploit binding assays, e.g.,an ELISA assay, receptor binding and nucleic acid probe hybridizationtechniques. Typically, with one microplate, e.g., micro-well titerplate, only one reading per well can be taken, e.g., by light emissionanalysis. These assays function in either one of two ways: (1)hybridization in solution; or (2) hybridization to a surface boundmolecule. In the latter case, only a single clement is immobilized perwell. This, of course, limits the amount of information that can bedetermined per unit of sample. Practical considerations, such as samplesize, labor costs, and analysis time, place limits on the use ofmicroplates in multiplex analyses. With only a single analysis,reaction, or determination per well, a multiple pathogen screen with theappropriate controls would consume a significant portion of a typical 96well format microplate. In the case where strain determination is to bemade, multiple plates must be used. Distributing a patient sample oversuch a large number of wells becomes highly impractical due tolimitations on available sample material. Thus, available patient samplevolumes inherently limit the analysis and dilution of the sample toincrease volume seriously affects sensitivity.

Polymerase Chain Reaction

Although, the polymerase chain reaction (PCR) can be used to amplify thetarget sequence and improve the sensitivity of the assay, there arepractical limitations to the number of sequences that can be amplifiedin a sample. For example, most multiplexed PCR reactions for clinicaluse do not amplify more than a few target sequences per reaction. Theresulting amplicons must still be analyzed either by Sanger sequencing,gel electrophoresis, or hybridization techniques such as Southernblotting or microplate assays. The sample components, by PCR's selectiveamplification, will be less likely to have aberrant results due to crossreactants. This will not be totally eliminated and controls should beemployed. In addition, PCR enhances the likelihood of false positiveresults from contamination, thus requiring environmental controls. PCRcontrols must also include an amplification positive control to ensureagainst false negatives. Inhibitors to the PCR process such ashemoglobin are common in clinical samples. As a result, the PCR processfor multiplexed analysis is subject to most of the problems outlinedpreviously. A high density of information needs to be acquired to ensurea correct diagnostic determination. Overall, PCR is not practical forquantitative assays, or for broad screening of a large number ofpathogens.

Probe-Based Hybridization Assays

Recently, probe hybridization assays have been performed in arrayformats on solid surfaces, also called "chip formats." A large number ofhybridization reactions using very small amounts of sample can beconducted using these chip formats thereby facilitating information richanalyses utilizing reasonable sample volumes.

Various strategies have been implemented to enhance the accuracy ofthese probe-based hybridization assays. One strategy deals with theproblems of maintaining selectivity with assays that have many nucleicacid probes with varying GC content. Stringency conditions used toeliminate single base mismatched cross reactants to GC rich probes willstrip AT rich probes of their perfect match. Strategies to combat thisproblem range from using electrical fields at individually addressableprobe sites for stringency control to providing separate micro-volumereaction chambers so that separate wash conditions can be maintained.This latter example would be analogous to a miniaturized microplate.Other systems use enzymes as "proof readers" to allow for discriminationagainst mismatches while using less stringent conditions.

Although the above discussion addresses the problem of mismatches,nucleic acid hybridization is subject to other errors as well. Falsenegatives pose a significant problem and are often caused by thefollowing conditions:

1) Unavailability of the binding domain often caused by intra-strandfolding in the target or probe molecule, protein binding, cross reactantDNA/RNA competitive binding, or degradation of target molecule.

2) Non-amplification of target molecule due to the presence of smallmolecule inhibitors, degradation of sample, and/or high ionic strength.

3) Problems with labeling systems are often problematic in sandwichassays. Sandwich assays, consisting of labeled probes complementary tosecondary sites on the bound target molecule, are commonly used inhybridization experiments. These sites are subject to the abovementioned binding domain problems. Enzymatic chemiluminescent systemsare subject to inhibitors of the enzyme or substrate and endogenousperoxidases can cause false positives by oxidizing the chemiluminescentsubstrate.

SUMMARY

The instant invention provides for both a multiplexed environment torapidly determine optimal assay parameters, as well as a fast,cost-effective, and accurate system for the quantitative analysis oftarget analytes, thereby circumventing the limitations of singledetermination assays. The optimization of a multiplexed assay can becarried out by experimental interrogation to determine the appropriatesolution conditions for hybridization and stringency washes. Thedevelopment of these optimal chemical environments will be highlydependent on the characteristics of the array of bound capture probemolecules, their complementary target molecules, and the nature of thesample matrix.

Multiplexed molecular analyses are often required to provide an answerfor specific problems. For example, determining which infectious agentout of a panel of possible organisms is causing a specific set ofdisease symptoms requires many analyses. Capture probe arrays offer theopportunity to provide these multivariate answers. However, the use ofsingle probe array platforms does not always provide enough informationto solve these kinds of problems. Recent innovative adaptations ofproximal charge-coupled device (CCD) technology has made it feasible toquantitatively detect and image molecular probe arrays incorporated intothe bottom of microplate wells. This creates a high throughput platformof exceptional utility, capable of addressing several applications withvery complex analysis parameters.

Uses

The multiplexed molecular analysis system of the instant invention isuseful for analyzing and quantifying several molecular targets within asample substance using an array having a plurality of biosites uponwhich the sample substance is applied. For example, this invention canbe used with microarrays in a microplate for multiplexed diagnostics,drug discovery and screening analysis, gene expression analysis, cellsorting, and microorganic monitoring (see examples below for each use).

Proximal CCD Imaging with Multiplexed Arrays

One application of the microplate based arrays of this invention is inparallel processing of a large number of samples. Large clinical labsprocess thousands of samples a day. A microplate configured with a fourby four (4×4) matrix of biosites in each of the 96 wells would be ableto perform a total of 1536 nearly simultaneous tests is from 96different patient samples utilizing the proximal CCD imager asillustrated in FIG. 1. FIG. 1 is a diagram showing a multiplexedmolecular analysis detection/imaging system. Moreover, a microplateconfigured with 15×15 arrays of probe elements in each of 96 wellsenables a total of 21,600 nearly simultaneous hybridization analyses,which becomes significant for analyzing gene expression from specificcells.

Throughput is also important when screening natural products forbiological activity. A matrix of biosites that model binding sites ofinterest may be placed in the bottom of each well and interrogated withan unknown product. Thousands of molecules may be screened per dayagainst these biosite arrays.

Creation of Hierarchical Arrays

Another use of the microplate based arrays is for the creation ofhierarchical arrays for complex analyses. In this format, multiplearrays operate in parallel to provide an answer to a complex assay. Theexample of the diagnostic assay provided in the Background sectionillustrates some of the parameters which should be considered in orderto provide an accurate result. For any specific analysis, a set of probeelements must be chosen. The selected probe elements should be able toselectively associate with defined targets without significant crossassociation to other macromolecules expected from either the patient orother organisms commonly associated with a specific sample type.Controls must be designed to prevent false positive or negative resultsfrom the sources outlined in the Background section. Once this is done,a combinatorial process can be used to identify the optimal associationand selectivity conditions for the defined analysis. For nucleic acidapplications, these conditions are highly dependent on the capture probelength and composition, target base composition, and sample matrix. Thenumber of arrays to be used depends on a number of different factors,e.g., the controls to be implemented and the differences in basecomposition of the capture probes. Ultimately, a set of integratedchemical devices emerge that can rapidly, efficiently, and accuratelyprovide an answer for the molecular analysis of interest.

Another use of the hierarchical arrays and the reaction vessel basedarrays would be for screening, samples for a broad range of possibletargets. In one case, a diagnostic test is performed to search for thecause of a defined set of symptoms. In most cases this narrows the rangeof possible organisms to a small number. Conversely, to screen donatedblood or tissue for a broader range of disease organisms, a decisiontree approach could be employed. Here an initial array or set of arrayscould be chosen to screen for broader classes of pathogens using probesfor highly conserved nucleic acid regions. Results from this wouldindicate which additional array sets within the microplate to samplenext, moving to greater and greater specificity. If enough sample isavailable, as might be the case with donated blood or tissue, all of thedecision tree elements could be interrogated simultaneously. If samplequantity is limiting, the approach could be directed in a serialfashion.

Assay development for any multiplex analysis is time consuming. Themicroplate based arrays as described herein can be used to speed theprocess for capture probe/target binding or hybridization. A definedarray can be deposited into each well of a microplate and then theassociation reactions are carried out using "gradients" of conditionsthat vary in two dimensions. For example, consider a 96 well microplatecontaining nucleic acids arranged in 8 rows by 12 columns. In one stepof the optimization, the effects of pH on various substrate compositionsmight be examined to see how this affects hybridization specificity.Twelve different pH's, one for each column, and 8 different surfacechemistries, one for each row could be used under otherwise identicalhybridization conditions to measure the effects on hybridization foreach capture probe/target element in the array. This type of analysiswill become essential as array technology becomes widely used and isamenable to any receptor/ligand binding type experiment.

The hierarchical array format, consisting of defined sets of arrays withindividually optimized chemical environments functioning in parallel toprovide an answer to a complex analysis, can be implemented in otherways. Instead of a batch process, where a series of analysis sets arepresent in each microplate, a hierarchical array analysis set can befashioned into a flow cell arrangement. This would be specific to aparticular analysis and consist of the appropriate array sets and thenecessary fluidics to take a single sample and deliver the appropriatealiquot to each array in the set. The fluidics will deliver theappropriate association and wash fluids to perform the reactions, asdefined for each array in the set.

Advantages

The multiplexed molecular analysis system of the instant invention hasmany advantages over the conventional systems. Some of these advantagesare discussed below.

High Throughput

Multiple DNA/RNA probe arrays can be fabricated in the bottom of 96 wellmicrotiter plates which offer the potential of performing 1,536 (96×16)to 21,600 (96×225) hybridization tests per microtiter plate. Each wellwill contain a probe array of N elements dispensed onto plastic or glassand bonded to the microtiter plate. Moreover, by coupling the microtitertrays to a direct (lensless) CCD proximal/imager, all 1,536 to 21,600hybridization tests can be quantitatively accessed within seconds atroom temperature. Such proximal CCD detection approach enablesunprecedented speed and resolution due to the inherently high collectionefficiency and parallel imaging operation. The upper limit to thehybridization tests per microtiter plate exceeds 100,000 based on a 100μm center-to-center spacing of biosites.

Low Cost

Since the capture probe volumes dispensed on the reaction substrate canbe limited to about 50 picoliters (pL), only 150 nanoliters (nL) ofcapture probe reagent is required to produce over 1,500 distinct bindingtests. The dispensing of the probe arrays on plastic rolls or on thinglass sheets can be efficiently performed in an assembly-line fashionwith a modular ink-jet or capillary deposition system.

Automated Operation

The multiplexed assay can be designed in a standard 96 well microtiterplate format for room temperature operation to accommodate conventionalrobotic systems utilized for sample delivery and preparation. Also, theproximal CCD-based imager with a graphical user interface will enablethe automation of the parallel acquisition of the numerous hybridizationtest results. The CCD imaging system software provides automatedfiltering, thresholding, labeling, statistical analysis and quantitativegraphical display of each probe/target binding area within seconds.

Versatility

The proximal CCD detector/imager utilized in a particular embodiment ofthe multiplexed molecular analysis system accommodates numerous moleculelabeling strategies including fluorescence, chemiluminescence andradioisotopes. Consequently, a single instrument can be employed for avariety of reporter groups used separately or together in a multiplexedmanner for maximal information extraction.

High Resolution

The accompanying proximal CCD detector/imager offers high spatial anddigital resolution. In the preferred embodiment, CCD pixel sizes ofapproximately 25×25 μm² will support the imaging of hundreds tothousands of individual biosites on a reaction substrate. Together with16 bit digital imaging, a highly quantitative image of the high densityof biosites is achieved.

Fast Time-to-Market

Since the approach outlined is based on previously demonstrated proximalCCD detection and imaging coupled with microarrays dispensed inconventional sized microtiter plates, the overall molecular analysissystem is expected to provide a fast time-to-market solution to complexmulticomponent molecular-based analyses.

Overall, the invention disclosed provides a method and apparatus forboth a multiplexed environment to rapidly determine the optimal assayparameters as well as a fast, cost-effective, and accurate system forthe quantitative analysis of molecules, thereby circumventing thelimitations of single determination assays.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing a multiplexed molecular analysisdetection/imaging system.

FIG. 2 is a printed computer image obtained with the proximal CCD imagershowing deposited DNA probe biosites with ink-jet printing.

FIG. 3 is a diagram showing the biosite deposition system usingstaggered ink-jet dispensing modules.

FIG. 4 is a diagram showing the biosite deposition system using multiplecapillaries.

FIG. 4a is a diagram showing biosite deposition with array templates.

FIG. 4b is a diagram showing biosite deposition into nanoliter wells.

FIG. 5a is a diagram illustrating the Universal Array concept.

FIG. 5b is a diagram showing direct binding for a target probeassociated with the Universal Array.

FIG. 5c is a printed computer image showing a multi-microtiter wellproximal CCD image of a 4×4 Universal Array.

FIG. 5d is a printed computer image showing a single microtiter wellproximal CCD image of a 4×4 Universal Array.

FIG. 6 is a diagram illustrating an ECL implementation in a reactionvessel with proximal CCD imaging.

FIG. 7 is a diagram showing fabrication of ECL reaction vessels.

FIG. 8 is a chemical drawing showing lanthanide chelators.

FIG. 9 is a diagram showing the electronics schematic of a multiplexedmolecular analysis system.

FIG. 10a is a diagram showing a CCD sensor array for the proximal CCDimager.

FIG. 10b is a diagram showing the tiling of CCD sensors to form a largeformat proximal CCD imager.

FIG. 10c is a diagram showing an alternative tiling scheme of multipleCCD sensors used to form a large format proximal CCD imager.

FIG. 11 is a printed computer image showing microarrays within amicroplate reaction vessel. One single reaction chamber is shown as aninsert.

FIG. 12 is a diagram showing glass and polypropylene surface couplingchemistries.

FIG. 13 is a diagram showing genotyping by universal point mutationscanning.

FIG. 14 is a diagram showing microtiter-based throughput genotyping.

FIG. 15 is a diagram showing homogeneous in situ microarray detection ofmultiplexed PCR amplicons.

FIG. 16 is a diagram showing homogeneous in situ microarray detection ofmultiplexed gap-ligase chain reaction products.

FIG. 17 is a diagram showing small molecule universal array (drugscreening/discovery).

FIG. 18 is a diagram illustrating spatial addresses of small moleculescovalently immobilized on amino-derivitized thin-bottom, glassmicrotiter wells.

FIG. 19A is a printed computer image showing specific imaging ofbiotin-addressable biosites detected using streptavidin:HRP conjugate(4×4 single well microarray).

FIG. 19B is a printed computer image showing specific imaging ofdigoxigenin-addressable biosites detected using anti-digoxigenin:HRPconjugate (4×4 single well microarray).

FIG. 19C is a printed computer image showing specific imaging offluorescein-addressable biosites detected using anti-fluorescein:HRPconjugate (4×4 single well microarray).

FIG. 19D is a printed computer image showing simultaneous imaging offluorescein, biotin, and digoxigenin biosites detected usinganti-fluorescein, anti-digoxigenin and streptavidin:HRP conjugates (4×4single well microarray).

DETAILED DESCRIPTION

Definitions

For the purpose of this invention, different words and phrases aredefined as follows:

By "target molecules or target analyte" is meant the molecules ofinterest in a substance which are to be interrogated by binding to thecapture probes immobilized in an array.

By "mRNA target molecule or mRNA target analyte" is meant a substancecontaining identical mRNA components or a mixture of disparate mRNAs.

By "capture probe, probe molecules or probes" is meant the moleculeswhich are deposited as biosites onto the reaction substrate forinterrogating the target molecules. Probes are meant to include nucleicacids, DNA, RNA, receptors, ligands, antibodies, anti-antibodies,antigens, proteins, and also small chemical compounds such as drugs,haptens, or peptides.

The term "hapten binding polypeptide" includes intact antibody moleculesas well as fragments thereof, such as Fab, F(ab')₂, Fv, single chainantibody (SCA), and single complementary-determining region (CDR). Forpurposes of the invention, "hapten" and "epitope" are consideredinterchangeable.

The term "array" refers to a two-dimensional spatial grouping or anarrangement.

By "hierarchical array" is meant an array arranged in an hierarchy orarranged in a graded or ranked series. Examples of different"hierarchical arrays" comprising the multiplexed assay of the inventioninclude, but are not limited to, an array of a 96 well microtiter plate,wherein there are N probe sites or biosites per well, wherein there are10⁷ to 10¹⁰ molecules for each probe site or biosite, wherein an arrayof M depositors are used to deposit probes in each probe site onto thefilm substrate that forms the bottom of the well in a 96 microtiter wellreaction chamber. The depositors can deposit the probes via manydifferent mechanisms, e.g., ink-jet deposition, capillary, andphotolithography.

The term "probe arrays" refers to the array of N different biositesdeposited on a reaction substrate which serve to interrogate mixtures oftarget molecules or multiple sites on a single target moleculeadministered to the surface of the array.

The term "oligonucleotide probe arrays" refers to probe arrays whereinthe probes are constructed of nucleic acids.

By "charge coupled device," also referred to as CCD, is meant awell-known electronic device which outputs an electrical signalproportional to the incident energy upon the CCD surface in a spatiallyaddressable manner.

The term "CCD proximal detection" refers to the use of CCD technologyfor detection and imaging in which the CCD is proximal to the sample tobe analyzed, thereby avoiding the need for conventional lenses.

By "ligands" is meant molecules which are recognized by a particularreceptor. "Ligands" may include, without limitation, agonists andantagonists for cell membrane receptors, toxins, venoms,oligosaccharides, proteins, bacteria and monoclonal antibodies.

By "multiplexed" is meant many or a multiple number.

By "multiplexed diagnostic assay" is meant a method for performing inparallel a large set or number of diagnostic assays. Thus a set ofparallel reactions can be handled with the same effort as a singlesample in previously described methods. Hence, a greater number ofassays can be handled within a fixed period of time. The parallel set ofreactions or multiplexed assay must be deciphered at the end of theprocess. This is done by labeling or tagging the biosite, as definedherein.

The term "reaction vessel" refers to an array of reaction chambers asdefined below. An example of a reaction vessel is a 96 well microtiterplate.

By "reaction chamber" is meant the environment in which thehybridization or other binding association takes place. Commerciallyavailable reaction vessels contain at least one reaction chamber, butcan contain 8, 24, 96 or 384 reaction chambers. For this invention,"reaction chamber(s)," "well(s)," "reaction site(s)," "reactionsubstrate(s)," "array hybridization site(s)," "hybridizationchamber(s)," and "hybridization well(s)," are used interchangeably. Anexample of a reaction chamber is one of the 96 microtiter wells in a 96well microtiter plate.

By "biosite" is meant the biological molecules or capture probes thatare deposited on the top surface of the reaction substrate, or basematerial. Under appropriate conditions, an association or hybridizationcan occur between the capture probe and a target molecule. The componentstrands of the biological molecule form the biosite since there is thepotential of a reaction occurring between each component strand of thebiological molecule and the target molecule. For example, each reactionchamber can contain at least one biosite. The maximum number of biositesper reaction chamber will depend on the size of the reaction vessel andon the practical optical resolution of the accompanying detector/imager.For example, an array of 16 (4×4 array) biosites may be deposited on thehybridization substrate or base material that eventually forms thebottom of the entire reaction vessel. Each biosite comprises a circle ofapproximately 25-200 microns (μm) in diameter. Thus, for a 16 biositearray, each of the 16×200 μm diameter area contains a uniform field ofprobes attached to the hybridization substrate (base material) in aconcentration which is highly dependent on the probe size and the wellsize. Each 25-200 μm diameter area can contain millions of probemolecules. Also, each of the 16 different biosites (probe sites) cancontain one type of probe. Thus, 16 different probe types can be assayedin an array containing 16 biosites (4×4 array) per reaction chamber. Asanother example, four separate 10×10 arrays (400 biosites) can begenerated to fit into one well of a 96 well microtiter plate withsufficient spacing between each of the 400 biosites. For this 10×10format, 400 hybridization experiments are possible within a singlereaction chamber corresponding to 38,400 (96×400) assays/hybridizationthat can be performed nearly simultaneously.

By "reaction substrate" is meant the substrate that the biosites orprobe sites are deposited on by using the depositors. Examples of"reaction substrates" include, without limitation, nylon membrane,polypropylene, polystyrene, vinyl, other plastics and glass.

By "modular deposition array" is meant an array of depositors. Thenumber of depositors depends primarily on the dimensions of the reactionsubstrate. For example, there can be four depositors fitted next to eachother, staggered regarding the front to back position of each depositor.Each depositor can be directly coupled to a housing reservoir. Thehousing reservoir holds a solution, e.g., a solution containing adesired probe at an appropriate concentration. The number of injectionmechanisms again depends on the design of the depositor, e.g., rangingfrom one to several injection mechanisms per depositor.

By "array formats on solid surfaces" is meant chip formats ormicroarrays.

By "throughput" is meant the number of analyses completed in a givenunit of time.

By "decision tree approach" is meant a sequential routing approach inwhich at each step an assessment is made which directs the subsequentstep.

By "hybridization detection" is meant to include, without limitation, ameans of two or more components to interact through hybridization, anassociation, linking, coupling, chemical bonding, covalent association,lock and key association, and reaction. For the purpose of thisinvention, these terms are used interchangeably.

By "methods of detecting (or detection) the association/hybridization"is meant to include, without limitation, fluorescent labeling,radioisotope labeling, chemiluminescence labeling, bioluminescencelabeling, colorimetric labeling. Labeling can be achieved by one of themany different methods known to those with skill in this art.

The term "luminescence" refers to, without limitation, electrical(electro), chemical, fluorescence, phosphorescence, bioluminescence, andthe like. However, for this invention, electrochemiluminescence orelectrical chemiluminescence (ECL) labeling is included as anothermethod of detection which does not require a wash step to remove excesstarget molecules from the solution, and is highly sensitive. For theelectrochemiluminescence or electrical chemiluminescence method ofdetection, once hybridization/association has occurred and a voltage hasbeen applied, only the labeled target molecules associated with thebiosite will emit light and be detected. The residual excess label inthe solution not associated with the biosite will therefore not emitlight.

This application is related to the following pending United StatesPatent Applications, incorporated herein by reference: U.S. Ser. No.07/794,036 entitled "Method and Apparatus for Molecular Detection" filedNov. 11, 1991, U.S. Ser. No. 08/353,957 entitled "Multiple MoleculeDetection Apparatus" issued Jul. 2, 1996, U.S. Ser. No. 08/457,096entitled "Multiple Molecule Detection Method" filed, Jun. 1, 1995, U.S.Ser. No. 07/872,582 entitled "Optical and Electrical Methods andApparatus for Molecule Detection" filed Apr. 23, 1992, U.S. Ser. No.08/511,649 entitled "Optical and Electrical Methods and Apparatus forMolecule Detection" filed Aug. 7, 1995, and U.S. Ser. No. 08/201,651entitled "Method and Apparatus for Detection and Imaging Particles"filed Feb. 25, 1994.

Overview

The multiplexed molecular analysis system of the invention can bedivided into four aspects:

A. Preparing the sample for subsequent association to a probe arraywithin the reaction chamber. This includes all front-end processes suchas purification, isolation, denaturation and labeling required toextract the target molecules from the sample.

B. Binding target molecules to the biosites within specialized reactionchambers in sufficient concentrations for association to occur.Following association, non-specific binding of target molecules is oftenminimized by washing out the reaction chambers.

C. Detecting and/or imaging the association (hybridization) of thetarget molecules with the biosites within each reaction chamber byproximal detection/imaging.

D. Processing the images to determine information about the targetmolecules such as the presence and amount of specific molecularconstituents within a given sample that leads to the analysis output.

The advantage of the instant invention lies in the particularimplementation of the above four procedures/steps, in particular in themethod and apparatus for:

STEP 1. Biosite Deposition. Biosite deposition relates to constructingmicroarrays.

STEP 2. Self Assembling Arrays--Universal Arrays. Creating andconstructing self assembling probe arrays or universal arrays enableson-line configuration of the biosites wherein an unvarying probe array(capture probes) is activated by binding to a cognate set of adapters(target probes) to yield a modified probe array which is specificallyconfigured for analysis of a target or target mixture. For thisinvention, "cognate" is defined for nucleic acids as a sequence which iscomplementary by the means of Watson-Crick pairing to another sequence.

STEP 3. Molecular Labeling Strategies. Molecular labeling strategiesrelates to versatile labeling of the target molecules (fluorescence,chemiluminescence, etc.) consistent with proximal large areadetection/imaging.

STEP 4. Detection System. A detection system relates to paralleldetection and/or imaging in the reaction vessel containing the reactionchambers using a proximal large area detector/imager.

Step 1--Biosite Deposition

Biosite deposition relates to constructing microarrays. There are manydifferent methods that may be used for depositing biosites into/onto thereaction chamber. Three of these approaches are taught below.

1. Ink-Jet Deposition

Ink-jet printing can be employed for printing the biological fluids toform the biosites. This approach provides very low droplet volumes (≈100pL with 75 μm diameter spot size) which minimizes reagents used andtherefore cost. Moreover, the printing process can be accelerated tothousands of droplets per second, thereby enabling a high throughputproduction capability for the reaction vessels.

One method useful for this invention utilizes electromechanically drivenink-jets which produce a volumetric change in the fluid by applying avoltage across a piezo-electric material (See Hansell, U.S. Pat. No.2,512,743, 1950). The volumetric changes causes a subsequent pressuretransient in the fluid which can be directed to form a drop on demand(D. Bogg et al., IBM Jour Res Develop (1984) 29:214-321.

Individual ink-jet devices can be integrated in a modular fashion toenable the printing of multiple fluids. For example, MicroFab Inc. hasdeveloped an ink-jet based DNA probe array printer constructed of eightmodular dispensing units, each with an integral 500 mL reservoirindependently addressed by respective drive and control electronics.

FIG. 2 depicts a printed computer image showing DNA probe biositesdeposited with ink-jet printing. FIG. 2 illustrates actual biositedeposition whereby an array of 100 DNA probe biosites per 1 cm² wasink-jet deposited onto a glass substrate. The array consists ofalternating columns of match and mismatch 12 mer probes which weresubsequently hybridized to a 12 mer single-stranded DNA target. Themismatch columns correspond to an A--A mismatch in the probe/targetcomplex. The actual image was captured within 1 second by the proximalCCD detector/imager described below.

L banks of modular ink-jet devices containing M depositors per modulecan be assembled in a staggered fashion to print L×M different biositeson the bottom surface of the reaction chambers as illustrated in FIG. 3.FIG. 3 is a diagram showing the biosite deposition system usingstaggered ink-jet dispensing modules. Here the reaction vessel is movedby a precision motor-controlled stage underneath the ink-jet devices forrapid printing. By constructing additional banks of modular ink-jetdevices and/or miniaturizing the individual depositors, an arbitrarilylarge number of distinct biosites can be printed in the reactionvessels. Alternatively, the printing can be performed on thin substratessuch as glass or plastics which are subsequently bonded to form thebottom of the reaction vessels.

2. Capillary Deposition

Another approach to biosite deposition involves the use of capillarytubing to dispense small amounts of the biosite solution onto thereaction substrate as illustrated in FIGS. 4, 4a, and 4b. FIG. 4 depictsa biosite deposition system using multiple capillaries. FIG. 4a is adiagram showing biosite deposition with array templates. FIG. 4b is adiagram showing biosite deposition into nanoliter wells. As shown, astorage vessel which contains the appropriate solutions is pressurizedmomentarily to prime tubes held in appropriate position by a manifold toinitiate the capillary dispensing action. With very small inner diametercapillaries (<50 μm), continuous pressure may be applied. Pressurepulses of varying duration can be utilized to deliver larger volumes ofsolution. Upon contact with the reaction substrate, the capillary tubessimultaneously deliver small volumes of the biosite solutions at preciselocations controlled by spatial arrangement of the bundled capillaries.

In this invention, the storage vessel allows for sampling either from astandard format microtiter plate or a customized plate designed to holdsmall volumes of liquid, allowing the capillary to efficiently dispensepicoliter volumes of liquid to many thousands of biosites with minimalloss to evaporation or possibility of cross contamination.

The lid of the storage vessel holder can be attached to a Z-axis motioncontrol device to allow for automated changes of the biosite solutionscontained in the microplates which may be delivered by a robotic arm.This is useful for printing sets of arrays containing large numbers ofsolutions, such as small molecule libraries used in drug discovery.

Also in this invention, the capillary tubing may be made of fused silicacoated with an outer layer of polyimide. These tubes are availablecommercially in any length with various widths and internal diameters.The preferred dimensions are 80 to 500 μm outer diameter (OD) and 10 to200 μm inner diameter (ID). The capillary bundles may be affixed to arobot arm and held in a precise pattern by threading the capillariesthrough array templates. An array template is a structure designed tomaintain the capillaries in the desired configuration and spacing, andmay consist of, without limitation, a metal grid or mesh, a rigidly-heldfabric mesh, a bundle of "sleeve" tubes having an inner diametersufficient to admit the fluid delivery capillaries, or a solid blockhaving holes or channels, e.g., a perforated aluminum block.

The embodiment depicted employs 190 μm OD capillaries, which arethreaded through an attachment site at the top of the printing fixture.The tubes extend down from the attachment site through an area thatallows for the capillaries to flex during printing. Below the flexregion the capillaries are threaded through an array template or a setof fused silica sleeves held in a grid pattern by the aluminum holderassemblies. The capillary sleeves/array template constitute an importantinnovation. The array templates/capillary sleeves also allow thecapillary tubing to travel smoothly and independently with respect toeach other in the vertical axis during printing.

The printing system can print high density probe arrays covering thebottom surface of microplate wells. To accomplish this, the printingsystem must be able to maintain a precise printing pattern andaccommodate irregular surfaces. Rigid tubes could be used to maintain aprecise pattern, however, they cannot readily accommodate irregularsurfaces. Flexible tubes will print on uneven surfaces but will notmaintain a precise printing pattern. The rigid sleeves, which extendbelow the aluminum holder assembly approximately 2 cm, support theflexible 190 μm OD fused silica capillary tubing and provide thestructural rigidity necessary to maintain a precise grid pattern overthis distance. The sleeves also allow the 190 μm tubing to travelsmoothly in the Z axis during printing. This ability coupled with theflexibility of the small OD capillary tubing allows for successfulprinting on surfaces that are not completely flat or absolutelyperpendicular to the printing fixture. Since the robot arm extends 0.1mm to 0.3 mm beyond the point where the capillary bundle contacts thesurface, the capillaries flex in the deflection zone illustrated in FIG.4 resulting in total surface contact among all capillaries in thebundle. When the printing fixture withdraws from the substrate, thecapillaries straighten, returning to their original positions. Thehighly parallel nature of the capillary bundle printing technique allowsfor microarrays containing from two to over 10,000 chemically uniquebiosites to be created with a single "stamp." The printer can printthese arrays at a rate of approximately one per second. This representsa greater than 10-fold increase in speed over existing technologies suchas photolithographic in situ synthesis or robotic deposition usingconventional load and dispense technology.

In photolithographic microarray synthesis, a series of masks aresequentially applied to build the nucleic acid probes a base at a time.An array of oligonucleotide probes each 12 bases long would require 48masks (12 nucleotide positions×4 bases). This process takesapproximately 16 hours to complete a wafer containing 48 microarrays.

Current robotic microarray printing or gridding systems are universallybased on various load and dispense techniques. These techniques can besplit into two categories. Active loading systems such as syringeneedles or capillaries draw up enough solution to dispense multiplebiosites or array elements before returning to reload or collect a newprobe solution. Pin style printing or gridding systems can only printone biosite per pin at a time. The pins are dipped into the probesolutions momentarily and the amount of solution adhering to the pin issufficient to print a single biosite. Both categories have limitationsthat are resolved by the capillary bundle printing system describedherein.

Production capacity is a primary constraint in microarray manufacturing,limiting the use of microarrays in high volume applications such as drugdiscovery due to the cost and limited availability. Forphotolithographic in situ synthesis, the constraint is the number ofindividual masks that must be applied to create an array of probes withthe necessary length to be effective. To increase capacity, theproduction systems must be duplicated. Current capacities for thisapproach (approximately 80,000 arrays for 1997) do not meet the needsfor the drug discovery market, where a single company may screen over100,000 samples per year.

Robotic printing systems currently manufacture microarrays in a largelyserial fashion. The geometry of the fluid reservoir is often responsiblefor the limited degree of parallel biosite deposition. This can beexplained by illustrating the process needed to produce a microarray. A"micro" array has a small overall dimension, typically smaller than 2 cmby 2 cm. The actual size is determined by the number of array elementsand their spacing, with an emphasis on reducing the overall size as muchas possible to reduce reagent costs and sample requirements. If aparallel printing approach is implemented using multiple pins ordepositiors, the geometries of these depositors must allow them tointerface with the probe solution reservoirs and still be able to fitwithin the confines of the area to be occupied by the microarray. If a 1cm² 100 element microarray (10×10) is to be constructed using a standard384 well microplate with wells spaced 4.5 mm on center as the probesolution reservoir, only 4 depositors can be used to printsimultaneously within the microarray. A total of 25 cycles of loadingand printing would be required to complete the array. In comparison,this array would be manufactured with a single print step for acapillary bundle printer with 100 capillaries. This is 50 times fasterthan robotic depositors using a load and dispense technique. If the samearray is condensed into a 0.5 cm² area, then only one depositor can beused, resulting in a 200-fold differential in manufacturing timecompared with the capillary bundle printer.

An important feature of the capillary bundle printer is the manner inwhich it interfaces to the printing solution storage vessel. Thecapillary bundles have a printing (distal) end and a storage vessel end.The printing solution is held in a sealed container that positions everycapillary in the printing bundle via a manifold so that each capillarydips into a specific well (supply chamber) of a microtiter plate, onecapillary per well. Current multi-well microtiter plates are availablewith 96, 384, or 1536 wells, and can contain up to 96, 384, or 1536individual probe solutions, respectively. For microarrays containingmore probe elements, multiple printing solution reservoirs or storagevessels can be interfaced to a single print head, as illustrated in FIG.4a. This design concept eliminates the geometry problems associated withload and dispense systems. The flexible fused silica capillaries can begathered together with the array templates or sleeves to create a printhead with capillaries spaced as close as 200 μm center to center.

The enclosed printing solution storage vessel is purged with an inertgas during the priming step of the printing process, which also servesto maintain an inert environment for the probe solutions. Contaminationof the probe solutions is minimized because of the single direction offlow through the capillaries. The printing end does not dip into thereservoir after every print cycle as in the load and dispensetechniques. This is important with contact printing where the depositorstouch the surface of the chip or slide that will contain the microarray.These surfaces are chemically treated to interact or bind to the probesolutions. Residual reactive chemicals, or even dust and dirt could beintroduced into the probe solution supply chambers with load anddispense systems. Often, the solution to be printed is available inlimited quantity or is very expensive. This is often the case inpharmaceutical drug discover applications where small moleculelibraries, containing hundreds of thousands of unique chemicalstructures that have been synthesized or collected and purified fromnatural sources, are used in high throughput screens of as manypotential disease targets as possible. These libraries must be used asefficiently as possible. The amount of fluid that is required for eachprinting system varies depending on the design. Most require a minimumof 100 microliters (μL) and are able to print less than 1,000 slides,with a significant amount of solution lost to washing between printcycles. The capillary array printer requires only 3 μL with less than 1μL used for the initial priming. This volume of printing solution issufficient to print between 20,000 to 30,000 microarrays with eachcapillary dispensing 50 to 100 pL per array. Load and dispense systemsdeliver anywhere from 800 pL to several nL per array.

The highly parallel approach allows probe solution deposition in amicroarray geometry (less than 2 cm×2 cm) independent of the geometry ofthe probe solution storage vessel. This permits production of an entiremicroarray containing from 2 to >10,000 unique capture probes (biosites)in a single stamp of the print head.

The flexible fused silica tubing (or other suitable material such asglass, Teflon or other relatively inert plastic or rubber, or thin,flexible metal, such as stainless steel) originating at the printingstorage vessel, pass through a series of arraying templates or sleevesthat are held at specific locations in the print head. An attachmentsite holds the capillaries in a fixed position that does not generallyallow horizontal or vertical movement. The capillaries extend down fromthis anchor point through an open area ("flexation zone") and into a setof array templates or sleeves. These lower array templates or sleevesserve to hold the printing capillaries in a geometry that matches themicroarray to be printed. The array templates limit the lateral movementof the printing capillaries to preserve the correct printing pattern,while allowing unrestricted vertical movement of each printing capillaryindependently of each other. This feature allows the print head to printon slightly irregular or uneven surfaces. The print head moves downwardto contact the substrate that is to receive the probe solutions, afterthe initial contact, the downward movement continues (the distancedepends on the surface, from 100 μm to a few mm) to ensure that all ofthe printing capillaries contact the surface. The flexation zonepositioned between the attachment site (that is holding the capillariesfixed) and the array templates or sleeves allows each capillary to bendso as to accommodate the "overdrive" of the print head. When the printhead moves up away from the substrate, the printing capillariesstraighten out again.

The capillary bundle originates in an enclosure containing discretefluid supply chambers, such as the wells in a microtiter plate. Eachcapillary is inserted into a specific well, which usually contains aunique probe solution with respect to the other wells. The storagevessel can be momentarily pressurized to begin the fluid flow in all ofthe capillaries simultaneously to prime the printer. After priming,continuous flow of the probe solutions through the capillaries isthereafter facilitated by adjusting the head height ΔH (the verticaldistance from the upper fluid reservoir and the printing tips, as shownin FIG. 4), or by electro-osmotic or electrophoretic force (where thetubes, storage vessels, and reaction chambers are appropriately modifiedto maintain and modulate an electro-osmotic and/or electrophoreticpotential). The chamber can maintain an inert environment bypressurizing the chamber with an inert gas, such as nitrogen or argon.

Fluid volumes deposited at each biosite can be modified by adjusting thehead height, by applying pressure to the printing solution storagevessel, by changing the length or inner dimension of the printingcapillaries, or by adjusting the surface tension of the probe solutionor the substrate that is being printed.

The prime and continuous print with multiple capillaries preventscontamination of the probe solution that can occur with load anddispense systems, which must contact the surface and then return to theprobe solution to draw more fluid. The continuous printing of thecapillary bundle printer is extremely efficient and proves to be anenabling technique for applications that require the use of smallvolumes of probe solution. The small outer and inner diameters of theprinting capillaries allow for printing as many as 10,000 spots per μLfrom a total volume of less than 5 μL.

In an alternative embodiment, the capillary tubes may be essentiallyrigid tubes (e.g., stainless steel) mounted in flexible or movablefashion at the attachment site, and slidably held by an array template.In this embodiment, the plurality of capillary tubes can be pressedagainst a reaction substrate and "even up" at their distal ends bymoving lengthwise through the array template, thus accommodating unevendeposition surfaces.

3. Photolithography/Capillary Deposition

To increase the spatial resolution and precision of the capillarydeposition approach, a combined photolithographic chemical masking andcapillary approach is taught herein. The first photolithographic stepselectively activates the precise biosite areas on the reactionsubstrate. Once selective activation has been achieved, the resultingcapillary deposition results in uniform biosite distribution.

Many different substrates can be used for this invention, e.g., glass orplastic substrates. With glass substrates, the procedure begins bycoating the surface with an aminosilane to aminate the surface. Thisamine is then reacted with a UV sensitive protecting group, such as thesuccimidyl ester of α(4,5-dimethoxy-2-nitrobenzyl) referred to as"caged" succimidate. Discrete spots of free amine are revealed on thecaged succimidate surface by local irradiation with a UV excitationsource (UV laser or mercury arc). This reveals free acid groups whichcan then react with amine modified oligonucleotide probes. Such aprocess provides for local biosite modification, surrounded by substrateareas with a relatively high surface tension, unreacted sites.

When using plastic substrates, the procedure begins by coating aminatedplastic with an amine blocking group such as a trityl which is poorlywater soluble and hence produces a coating with high surface tension.Next, an excitation source (eximer or IR laser for example) is used toselectively remove trityl by light-induced heating. The biosite areasare then activated with bifunctional NHS ester or an equivalent. The netresult is similar for glass wherein the locally activated biosite areaswill have low aqueous surface tension which are surrounded by relativelyhigh surface tension, thereby constraining the capillary dispensing tothe biosite area.

Step 2--Self Assembling Arrays--Universal Arrays

Creating and constructing self assembling probe arrays or universalarrays enables on-line configuration of the biosites wherein anunvarying probe array (capture probes) is activated by binding to acognate set of adapters (target probes) to yield a modified probe arraywhich is specifically configured for analysis of a target or targetmixture.

The Universal Array format overcomes significant obstacles thatcurrently prevent probe array technology from being implemented in acommercially broad manner. Fundamentally, probe arrays that allow forhighly parallel analysis of binding events require specialized equipmentto manufacture and sophisticated instrumentation to interpret thebinding patterns. Unfortunately, the current manufacturing processes formaking biosite arrays, such as ink-jet, robotic deposition, orphotolithographic in-situ synthesis are relatively inflexible. Thesetechniques are designed to make a large number of specific arrays tocover the cost of setup and operation. Hence, small volume custom arrayswould be prohibitively expensive.

In contrast, the Universal Arrays system as taught herein solves thisproblem by taking advantage of efficient high volume manufacturingtechniques for the capture probe arrays only. In this fashion, eachcustomer can use a pre-manufactured, high density biosite capture arraythat is readily "tailored or customized" by the end-user for theirspecific target analyte screening. For this invention, "target analyte"is defined as the solution-state solute to be analyzed via binding tothe probe array. In short, customization of the array can be performedin the customer's laboratory. The end-user synthesizes or producesbifunctional target probes containing two separate binding domains, onebinding domain cognate to a specific member in the array (capturedomain) and another binding domain specific for the target analytes ofinterest (target domain). In an actual assay, end-users add theircustomized bifunctional probes to a solution phase mixture of analytesand incubate in a reaction chamber containing a pre-manufactureduniversal capture array. Alternatively, the capture array is incubatedfirst with the bifunctional probes followed by an addition of theanalyte mixture (see FIG. 5). FIG. 5a is a diagram showing a UniversalArray. The analytes self assemble onto the array in a sandwich-mode byselective binding of their bifunctional probes to both the complementaryportions of the target and the capture array. The resulting addressable,self assembled arrays is easily analyzed with the complimentary proximaldetector/imager. Teachings for constructing the surface bound captureprobes and target probes are outlined below.

1. Capture Probes

The surface bound universal capture probes are arranged in an array ofbiosites attached to a solid support. Each biosite consists of amultitude of specific molecules distinct in function or composition fromthose found in every other biosite in the array. These capture probesare designed to have a specific composition or sequence to provide rapidand efficient binding to the capture domain of the target probes. Thespecific composition is also chosen to minimize cross associationbetween capture probes and their specific target probes.

Specifically for a nucleic acid capture probe the surface bound capturearray should be designed for optimum length, base composition, sequence,chemistry, and dissimilarity between probes.

The length of the nucleic acid capture probe should be in the range of2-30 bases and preferably in the range of 5-25 bases. More preferably,the length ranges from about 10-20 bases and most preferably is at orabout 16 bases in length to allow for sufficient dissimilarity amongcapture probes. Length is also adjusted in this range to increase targetprobe binding affinity so that capture probe arrays can be activated byaddition of target probe mixtures as dilute as 10⁻⁹ M. This allowstarget probes to be synthesized in small scale and inexpensively. Also,length is adjusted to this range to reduce the rate of target probedissociation from capture probe arrays. This allows the activatedcapture probe arrays to be washed thoroughly to remove unbound targetprobes, without dissociation of specifically bound target probes fromthe surface. With capture probes in such a size range, the complexformed by and between the target probe and capture probe interaction isstable throughout subsequent air drying, and can be stored indefinitelywith refrigeration.

A preferred percentage base composition for capture probe array sets isin the range of at or around 30-40% G, 30-40% C, 10-20% A, 10-20% T.Relatively G+C rich capture probes are desirable such that thethermodynamic stability of the resulting capture/target probe pairingwill be high, thus allowing for surface activation at low added targetprobe concentrations (e.g., in the range of 10⁻⁹ M). Nearest neighborfrequency in the capture probe set should minimize G-G or C-C nearestneighbors. This criterion minimizes the possibility of side reactions,mediated via G-quartet formation during capture probe attachment to thesurface, or during the capture probe-target probe binding step.

For capture probe sets it is desirable to obtain a set structure suchthat each member of the capture probe set is maximally dissimilar fromall others. To obtain such maximally dissimilar sets, the followingalgorithm can be employed.

1) The set size is defined. In a preferred embodiment, 16, 24, 36, 48,49, 64, 81, 96 and 100 constitute useful sizes.

2) The overall sequence structure of the capture probe set is defined.The length and base composition as described above are used to definesuch parameters. In general, the number of G bases and C bases are heldequal as are the number of A bases and T bases. This equality optimizesthe configurational diversity of the final sets. Thus, such sets will bedescribed by the equation G_(n) C_(n) A_(m) T_(m).

3) For a set structure defined by m and n, a random number generator isemployed to produce a set of random sequence isomers.

4) One member of the random sequence set is selected to be used aselement #1 of the set.

5) The maximum similarity allowable among set members is defined.Similarity is defined in terms of local pair-wise base comparison. Forexample, when two oligomer strands of identical length n are alignedsuch that 5' and 3' ends are in register, the lack of mismatches refersto the situation where at all positions 1-n, bases in the two strandsare identical. Complete mismatching refers to the situation wherein, atall positions 1-n, bases in the two strands are different. For example,a useful maximum similarity might be 10 or more mismatches within a setof 16, 16 mer capture probes.

6) A second member of the random sequence set is selected and itssimilarity to element #1 is determined. If element #2 possesses lessthan the maximum allowable similarity to element #1, it will be kept inthe set. If element #2 possesses greater than the maximum allowablesimilarity, it is discarded and a new sequence is chosen for comparison.This process is repeated until a second element has been determined.

7) In a sequential manner, additional members of the random sequence setare chosen which satisfy the dissimilarity constraints with respect toall previously selected elements.

Standard deoxyribonucleic acid base homologues, or homologues withmodified purine or pyrimine bases, or modified backbone chemistries suchas phophoramidate, methyl phosphonate, or PNA may be employed insynthesis of capture probes.

The capture probe should be linked to a solid support. This can be doneby coupling the probe by its 3' or 5' terminus. Attachment can beobtained via synthesis of the capture probe as a 3' or 5' biotinylatedderivative, or as a 3'/5' amine modified derivative, a 3'/5'carboxylated derivative, a 3'/5' thiol derivative, or as a chemicalequivalent. Such end-modified capture probes are chemically linked to anunderlying microtiter substrate, via interaction with a streptavidenfilm (for biotin), coupling to surface carboxylic acids or epoxidegroups or alkyl halides or isothiocyanates (for amines) to epoxides oralkyl halides (for thiols) or to surface amines (for carboxylic acids).Other attachment chemistries readily known to those skilled in the artcan be substituted without altering general performance characteristicsof the capture probe arrays. Capture probe arrays can be fabricated bysuch chemistries using either robotic or micro ink jet technology.

In order to minimize cross hybridization during the target probeactivation step, capture probe sets are constructed such that everymember of the capture probe set has a length which is identical ordiffers by no more than 1 base from the average length of the set, andpossesses an overall gross base composition which is identical orsubstantially similar to all other members of the set. These twocriteria interact to allow the free energy of all target probe/captureprobe pairings to be identical. The above described algorithm generatessuch sets of probes.

It is important that the sequence of each member of the capture probeset differ from every other member of the capture probe set by at least20%, preferably 40%, more preferably 50% and most preferably 60%. Thisextent of sequence homology (less than 80% between any two members ofthe set) prohibits target probes from binding to members of the probeset other than that to which it has been designed.

There are numerous capture probe sets that satisfy the general designcriteria as outlined above. Presented below is a specific example of a16 element capture probe set generated by the above described algorithmwhich adequately satisfies the above criteria.

For this example, capture probe length is held at 16 bases and basecomposition is fixed at G₅ C₅ T₃ A₃ among all 16 members of the set.There are no more than 3 G-G or C-C pairings per capture probe element.This particular capture probe set is designed to be linked to microtitersupport via an amine linkage at its 3' terminus. However, a 5' aminelinkage, or other chemistries could have been used as well.

The top-most array element (#1) has been chosen as a standard. Detailedinspection of this set shows that every member of the set differs fromevery other member of the set by at least 10 base mismatches, thussatisfying the criterion of no more than 50% homology between captureprobe set elements.

    SEQUENCE#      CAPTURE PROBES, 16 MERS                                        ______________________________________                                          SEQ ID NO:1 5'-TGATTCAGACCGGCCG-3'a                                            - SEQ ID NO:2 5'-CCCGGGGCGTCTTAAC-3'a                                         - SEQ ID NO:3 5'-GGACGCCATATGCGCT-3'a                                         - SEQ ID NO:4 5'-TGAGGGCTCCGCCATA-3'a                                         - SEQ ID NO:5 5'-AACCCGTGACGTGTGC-3'a                                         - SEQ ID NO:6 5'-AGCATCGCCGGTCCTG-3'a                                         - SEQ ID NO:7 5'-CCTGCAAGGCTGACGT-3'a                                         - SEQ ID NO:8 5'-CAGTTGTCGACCCCGG-3'a                                         - SEQ ID NO:9 5'-CGGCGCGTCCAATTCG-3'a                                         - SEQ ID NO:10 5'-ATCGATCTGAGGGCCC-3'a                                        - SEQ ID NO:11 5'-GTACATGCGGCCTGCA-3'a                                        - SEQ ID NO:12 5'-TAGCCGCTCGCTAGAG-3'a                                        - SEQ ID NO:13 5'-CCTAGTGATGACCGGC-3'a                                        - SEQ ID NO:14 5'-GTCTGAGGGCAACCTC-3'a                                        - SEQ ID NO:15 5'-CTAGCTGGCTACGCAG-3'a                                        - SEQ ID NO:16 5'-GCCATCCGCTTGGAGC-3'a                                     ______________________________________                                         a = amine linkage to solid support, such as a 3' propanolamine, coupled t     a carboxylate modified surface via amide linkage or epoxide modified          surfaces.                                                                

                    ELEMENTAL TARGET PROBES - SEQUENCE# (cognate to capture                      probes)                                                        ______________________________________                                          SEQ ID NO:17 3'-TTACTAAGTCTGGCCGGC-5'                                          - SEQ ID NO:18 3'-TTGGGCCCCGCAGAATTG-5'                                       - SEQ ID NO:19 3'-TTCCTGCGGTATACGCGA-5'                                       - SEQ ID NO:20 3'-TTACTCCCGAGGCGGTAT-5'                                       - SEQ ID NO:21 3'-TTTTGGGCACTGCACACG-5'                                       - SEQ ID NO:22 3'-TTTCGTAGCGGCCAGGAC-5'                                       - SEQ ID NO:23 3'-TTGGACGTTCCGACTGCA-5'                                       - SEQ ID NO:24 3'-TTGTCAACAGCTGGGGCC-5'                                       - SEQ ID NO:25 3'-TTGCCGCGCAGGTTAAGC-5'                                       - SEQ ID NO:26 3'-TTTAGCTAGACTCCCGGG-5'                                       - SEQ ID NO:27 3'-TTCATGTACGCCGGACGT-5'                                       - SEQ ID NO:28 3'-TTATCGGCGAGCGATCTC-5'                                       - SEQ ID NO:29 3'-TTGGATCACTACTGGCCG-5'                                       - SEQ ID NO:30 3'-TTCAGACTCCCGTTGGAG-5'                                       - SEQ ID NO:31 3'-TTGATCGACCGATGCGTC-5'                                       - SEQ ID NO:32 3'-TTCGGTAGGCGAACCTCG-5'                                    ______________________________________                                    

2. Target Probes

A target probe set is designed and constructed to bind to the captureprobe set in a specific manner, i.e., each target probe element binds toonly one element of the capture probe set. Thus, a mixture of targetprobes can be administered to a capture probe array formed on the bottomof a microtiter well, or equivalent surface. For the nucleic acidembodiment of the Universal Array, subsequent to binding, the targetprobe set will partition itself among capture probe set members viaWatson-Crick base pairing, thereby delivering a unique binding domain(cognate to analyte) to each site in the probe array.

There are two general methods that can be employed by the end-user tosynthesize customized nucleic acid-based bifunctional target probes. Thesimplest and most direct method is to synthesize a singleoligonucleotide that contains the two domains (capture and analyte)separated by a linker region using a standard automated DNA synthesizer.As a class, the bifunctional target probes for a nucleic acid embodimentpossess a structural domain cognate to the capture probe which is theWatson-Crick complement to one element of the capture probe set. Itslength and base sequence is thus defined by that of the capture probevia standard rules of antiparallel Watson-Crick duplex formation. Inaddition, the target probe also contains one of the following structuraldomains:

a. Cognate to a Small Segment of a Solution State Nucleic Acid TargetAnalyte

This is the component of the target probe which is complementary viaWatson-Crick pairing to the solution state target nucleic acid to beanalyzed. In general, its sequence has no correlation to that of thedomain which is cognate to the capture probe. However, several generaldesign criteria should be met.

First, for ease of target probe synthesis, the unique domain in therange of about 5-30 bases in length, and preferably in the range ofabout 10-25 bases in length. With shorter target probe domains, analytebinding affinity is insufficient, and longer target probe domainspresent synthesis difficulties.

Second, when the unique sequence is equal in length or longer than thecapture probe set, the unique element should possess a sequence which isno more than 80% homologous to the Watson-Crick complement of anycapture probe element. This criterion eliminates inappropriateassociation of the unique target probe segment with members of thecapture probe set.

b. Cognate to a Priming Site Used for Biochemical Amplification Such asPCR and LCR

This domain essentially creates nucleic acid amplification primers withtails complementary to capture probe sites in a Universal Array. Afteramplification, the resulting amplicon sets can be directly hybridized tothe capture probe array and analyzed as described below.

C. Chemically Modified for Direct Linkage

Another method of synthesizing bifunctional DNA target probes consistsof individually and separately synthesizing analyte and capture sequenceoligos that are chemically altered to incorporate a reactivefunctionality which will allow subsequent chemical linkage of the twodomains into a single bifunctional molecule. In general, the 5' or 3'terminus of each oligo is chemically altered to facilitate condensationof the two sequences in a head to tail or tail to tail manner. A numberof methods are known to those skilled in the art of nucleic acidsynthesis that generate a variety of suitable functionalities forcondensation of the two oligos. Preferred functionalities includecarboxyl groups, phosphate groups, amino groups, thiol groups, andhydroxyl groups. Further, chemical activation of these functionalitieswith homo- or heterobifunctional activating reagents allows forcondensation of the activated oligo with the second functionalized oligosequence. Some examples of the various functionalities and activatingreagents that lead to condensation are listed below:

    ______________________________________                                        Terminal Functionality                                                                       ACTIVATING AGENT                                                 (3' or 5') (Homo or Heterobifunctional)                                     ______________________________________                                        NH.sub.2  (amino)                                                                            NHS--NHS, NHS-maleimide, iodoacetic                               anhydride, EDC (carbo-diimide)                                               SH.sub.2  (thiol) maleimide-NHS                                               COOH (carboxyl) EDC (carbodiimide)                                            OH (hydroxyl) carbodiimide (EDC)                                              PO.sub.4  (phosphate) N-methylimidazole (EDC)                                 PO.sub.3 S alpha-thiophosphate maleimide-maleimide, maleimide-NHS           ______________________________________                                    

A specific example of the target probe domains that are cognate to thecapture probe set of the Universal Array and can be modified to allowfor direct binding to a specifically modified probe, nucleic acid orother molecule capable of selective binding to the analyte of interestis illustrated in FIG. 5b. FIG. 5b is a diagram showing direct bindingfor a target probe. As shown in FIG. 5b, the target probe is constructedfrom two parts; the first is a presynthesized probe (TP1) complementaryto a capture probe which has a linkage element for attaching the secondtarget complex (TP2). Such embodiment yields a high degree of simplicityfor the customer since the first target component can be offered in aready-to-use format.

A sample protocol for the two piece approach is as follows:

1) Obtain TP1 from commercial source, e.g., Genometrix (synthesized as3' amine, 5' thiol);

2) TP2 synthesized as an amine;

3) TP2 is mixed with iodoacetic acid anhydride in "Buffer A" to generatethe iodoacetate derivative TP2*;

4) Ethanol ppt, run over G25 spin column and collect the excluded volumewhich contains TP2* only, with small molecule reactants removed;

5) TP1+TP2* are mixed with "Buffer B";

6) Separate on G50 spin column.

For this invention, "Buffer A" consists of 10 mM sodium citrate, pH 7.0,and "Buffer B" consists of 10 mM sodium bicarbonate, pH 9.0.

3. Linker

In some instances, a chemical linker may be needed to separate the twonucleic acid domains of the target probe, to minimize stearicinteraction between the target probe and the solution state nucleic acidanalyte. This linker may be constructed from nucleic acid buildingblocks. For example, the sequence T_(n) (where n=1-5) is preferredbecause stretches of T are readily synthesized and minimize thelikelihood of sequence dependent interactions with capture probe, othertarget probe domains, or the solution phase nucleic acid analyte.

However, the linker is more preferably synthesized from an inertpolymer, such as oligo-ethylene glycolate linkages(--O--CH2--CH2--O--)_(n). Linkages with n=3 are commercially availableas the phosphoramiodate for ready synthesis into oligonucleic acids viastandard phosphodiester linkages. From one to five linkers canintroduced as needed.

Detailed below is a specific example of the invention based upon thecapture probe set described above. Here, the linker domain is listed astwo repeats of a triethylene glycolate synthon, linked by aphosphodiester linkage into the target oligonucleotide backbone.

           NUCLEIC ACID ANALYTE(TP2)     CAPTURE PROBE(TP1)                         5'-----CCACACTGGAACTGAGA------3' 5'-TGATTCAGACCGGCCG-3'a                             IIIIIIIIIIIIIIIII            IIIIIIIIIIIIIIII                              3'-GGTGTGACCTTGACTCT-----(Tn)---ACTAAGTCTGGCCGGC-5'                               TARGET PROBE 2   T LINKER  TARGET PROBE 1                              -      NUCLEIC ACID ANALYTE            CAPTURE PROBE                         5'-----CCACACTGGAACTGAGA------3'  5'-TGATTCAGACCGGCCG-3'a                            IIIIIIIIIIIIIIIII             IIIIIIIIIIIIIIII                             3'-GGTGTGACCTTGACTCT-----(X)-----ACTAAGTCTGGCCGGC-5'                              TARGET PROBE 2   X LINKER    TARGET PROBE 1                            - X = -OPO.sub.2 -{O-CH.sub.2 -CH.sub.2 -O-OPO.sub.2 -].sub.2 -O                                                  DIETHYLENE GLYCOLOATE LINKAGE                                                   - TARGET PROBE 1                       3'AMINE-----------5'-OPO.sub.2 -O-CH.sub.2 -CH.sub.2 -CH.sub.2 -CH.sub.2                                            -CH.sub.2 -CH.sub.2 -SH                  -           3' PROPYL AMINE          TARGET PROBE 2                                  NH2-CH.sub.2 -CH(OH)-CH2-OPO.sub.2 -].sub.2 -O-3'-------------5'       -      IODO ACETATE DERIVATIVE        TARGET PROBE 2                         I-CH2-CO-NH-CH.sub.2 -CH(OH)-CH2-OPO.sub.2 -].sub.2 -O-3'-------------5'       -                   COUPLED TP1 + TP2 PRODUCT                                 -      5' THIOL DERIVATIVE OF TP1       3' ALKYL HALIDE OF TP2                                                      - 3'-[TP1]-OPO.sub.2 -O-CH.sub.2                                            -CH.sub.2 -CH.sub.2 -CH.sub.2                                                 -CH.sub.2 -CH.sub.2 -S-CH.sub.2                                               -CO-NH-CH.sub.2 -CH(OH)-                 CH.sub.2 -OPO.sub.2 -].sub.2 -O-[TP2]-5'                                       - TP1 = target probe 1                                                       TP2 = target probe 2                                                    

A Universal Array having 16 capture probes within a single well of a 96well microtiter plate is shown in FIG. 5c. FIG. 5c is a printed computerimage showing a multi-microtiter well proximal CCD image of a 4×4Universal Array. In FIG. 5c, target specific hybridization is observedin 15 out of the 16 oligo elements in the array. The results of 15target specific hybridizations conducted simultaneously in 3 separatereaction chambers in a multiwell reaction vessel are quantitativelyassessed from the digital image obtained from the proximal CCD imager.Hybrids are digoxigenin end-labeled oligonucleotide targets detectedusing anti-digoxigenin antibody-alkaline phosphatase conjugate and ELF™fluorescence. In this assay (from Molecular Probes, Inc.) the antibodybinds to the digoxigenin group, delivering alkaline phosphatase to thebound target. The alkaline phosphatase converts the non-fluorescent ELFprecursor to a fluorescent product which can be detected by UVirradiation.

FIG. 5d is a printed computer image showing a single microtiter wellproximal CCD image of a 4×4 universal array. FIG. 5d shows the targetspecific hybridization of 4 of the 16 oligonucleotide elements in thearray at positions A2, B2, C2, and D2. Note the desirable absence ofsignificant cross hybridization, which has been specifically minimizedby imposing the maximum dissimilarly design constraints. Hybrids aredigoxigenin end-labeled oligonucleotide targets detected usinganti-digoxigenin alkaline phosphatase conjugate and ELF™ fluorescence asdescribed above.

4. Non-Nucleic Acid Embodiments

Small molecule Universal Arrays can be employed for rapid, highthroughput drug screening. In this format, surface bound capture probesconsist of small haptens or molecules arranged in separated biositesattached to a solid support. Each biosite consists ofspecifically-addressable, covalently immobilized small molecules such ashaptens, drugs and peptides. These organic capture molecules aredesigned to have a high affinity association with a bispecific ligand.These ligands contains both a domain cognate to the small immobilizedorganic molecule (capture probe) and cognate to the analyte of interest.The domain cognate to the analyte can associate either directly to thistarget or to a label on the analyte.

Specific examples of bispecific ligands include, without limitation,antibody:antibody, antibody:receptor, antibody:lectin,receptor:receptor, bispecific antibodies, antibody:enzyme,antibody:streptavidin, and antibody:peptide conjugates.

Analytes can include, but are not limited to, dsDNA, ssDNA, total RNA,mRNA, rRNA, peptides, antibodies, proteins, organic enzyme substrates,drugs, pesticides, insecticides and small organic molecules.

Conversely, the format for a small molecule Universal Array can beinverted so that the macromolecular ligand becomes the capture probe.Thus, a Universal Array (Macromolecular Universal Array) may containlarge macromolecules such as, without limitation, antibodies, proteins,polysaccachrides, peptides, or receptors as the immobilized captureprobe. In turn, unique small molecule tags having a specific, highaffinity association for the macromolecular biosites are covalentlyattached to various probes cognate to the analyte. These labeled probesnow represent the bispecific component cognate to both the capturemacromolecule and the target analyte. Some representative examples ofsmall molecules (haptens or drugs) are listed in Table 1 below. This isonly a partial list of commercially available antibodies to haptens,steroid hormones and other small molecule drugs. Examples of thesebispecific, small molecule-labeled macromolecules include antibodies,receptors, peptides, oligonucleotides, dsDNA, ssDNA, RNA,polysaccharides, streptavidin, or lectins. A partial list of 48representative compounds for which specific antibodies are availableinclude: fluorescein; dinitrophenol; amphetamine; barbiturate;acetaminophen; acetohexamide; desipramine; lidocaine; digitoxin;chloroquinine; quinine; ritalin; phenobarbital; phenytoin; fentanyl;phencyclidine; methamphetamine; metaniphrine; digoxin; penicillin;tetrahydrocannibinol; tobramycin; nitrazepam; morphine; Texas Red;TRITC; primaquine; progesterone; bendazac; carbamazepine; estradiol;theophylline; methadone; methotrexate; aldosterone; norethisterone;salicylate; warfarin; cortisol; testosterone; nortrptyline; propanolol;estrone; androstenedione; digoxigenin; biotin; thyroxine; andtriiodothyronine.

The general concept of Universal Arrays, whether they be DNA-based,small molecule-based, or protein-based allows for great versatility andend-user friendliness. The various configurations described allow forhighly parallel, simultaneous, multiplexed, high throughput screeningand analysis of a wide variety of analyte mixtures.

Step 3--Molecular Labeling Strategies

Molecular labeling strategies relate to versatile labeling of the targetmolecules (fluorescence, chemiluminescence, etc.) consistent withproximal large area detection/imaging.

1. Introduction--Conventional Labeling

Labeling can be achieved by one of the many different methods known tothose skilled in the art. In general, labeling and detection of nucleicacid hybrids may be divided into two general types: direct and indirect.Direct methods employ either covalent attachment or direct enzymaticincorporation of the signal generating moiety (e.g., isotope,fluorophore, or enzyme) to the DNA probe. Indirect labeling uses ahapten (e.g., biotin or digoxigenin) introduced into the nucleic acidprobe (either chemically or enzymatically), followed by detection of thehapten with a secondary reagent such as streptavidin or antibodyconjugated to a signal generating moiety (e.g., fluorophore or signalgenerating enzymes such as alkaline phosphatase or horseradishperoxidase).

For example, methods of detecting the association/hybridization include,without limitation, fluorescent labeling, radioisotope labeling,chemiluminescence labeling, bioluminescence labeling, colorimetriclabeling and electrochemiluminescence labeling. Many known labelingtechniques require a wash step to remove excess target from thehybridization/association solution, e.g., fluorescent, radioisotope,chemiluminescence, bioluminescence and colorimetric labeling. Several ofthese will be described below.

2. Fluorescent Labeling

Fluorescent labeling is suitable for this invention for several reasons.First, potentially hazardous substances such as radioisotopes areavoided. Furthermore, the fluorescent labeling procedures are simplerthan chemiluminescent methods since the latter requires enzymaticreactions and detection in the solution state. Finally, the fluorescentlabeling approach can be modified to achieve the highest signal-to-noseratio SNR among the safest labeling techniques by utilizing secondarylinker chemistries that enable the attachment of hundreds of fluorescentdye molecules per target molecule.

The particular fluorescent dyes to be considered include commerciallyavailable agents such as ethidium bromide, as well as the novel dyesproposed in the affiliated chemistry component. These labeling agentshave intense absorption bands in the near UV (300-350 nm) range whiletheir principle emission band is in the visible (500-650 nm) range ofthe spectrum. Hence, these fluorescent labels appear optimal for theproposed proximal CCD detection assay since the quantum efficiency ofthe device is several orders of magnitude lower at the excitationwavelength (337 nm) than at the fluorescent signal wavelength (545 nm).Therefore, from the perspective of detecting luminescence, thepolysilicon CCD gates have the built-in capacity to filter away thecontribution of incident light in the UV range, yet are very sensitiveto the visible luminescence generated by the proposed fluorescentreporter groups. Such inherently large discrimination against UVexcitation enables large SNRs (greater than 100) to be achieved by theCCDs.

3. Electrochemiluminescence Labeling

Electrochemiluminescence or electrical chemiluminescence (ECL) labeling,e.g., ruthenium (Ru) does not require a wash step to remove excesstarget from the solution and is highly sensitive. Briefly, forelectrochemiluminescence as a method of detection, the internal surfaceof the reaction chamber is coated with a conductive material, e.g.,gold, and the biosite is attached to this conductive surface (See FIG.6). FIG. 6 is a diagram showing ECL implementation in reaction vesselwith proximal CCD imaging. Using one microtiter well (of a 96 microtiterwell plate) as a reaction chamber, the biosites are deposited onto theinternal circumference of the microtiter well by one of several methodsas described above (ink-jet, capillary, or photolithography/capillary).

This conductive surface acts as a cathode (positive lead), and an anode(negative lead) is provided by inserting a metal cup with an electrodeprotruding through its center into the reaction chamber (microtiterwell). The electrode is positioned such that it is inserted into thehybridization solution. The voltage applied to the anode induces anelectrochemical event at the labeled molecule surface which releasesenergy in the form of photons (light).

The specific ECL label, e.g., Ru, is attached to the target molecule bythe conventional means. The labeled target is added to the hybridizationsolution and once hybridization occurs between the Ru labeled target andbiosite, e.g., after sufficient time has passed for hybridization to becompleted, a voltage is applied and only Ru labeled target associated(hybridized) with the biosite will emit light and be detected. In orderfor the Ru labeled target to be detected, it must be in proximity to thecathode. The residual excess Ru labeled target not associated with thebiosite will therefore not emit light.

The ECL reaction vessel is diagramed in FIG. 7. In FIG. 7, the thin filmsubstrate, e.g, plastic, glass, etc., is patterned with a conductivemetal, e.g., gold, platinum, etc., to form electrodes within thereaction chambers. Next, the biosites are deposited with one of severalmethods described above (ink-jet, capillary,photolithographic/capillary) onto the patterned electrodes. Finally, theresulting thin film substrate is bonded onto the reaction vessel whichserves as the bottom of the reaction chambers.

4. Lanthanide Chelate Labeling

As an alternative to ethidium-based fluorescent reporter groups, whichare known for their tendency to absorb nonspecifically to surfacescausing increased signal background, the use of aromatic lanthanide (Ln)chelators may be used in the instant invention. Although the lanthanideions (Tb and Eu specifically) have luminescent yields near to one (1),and emission lifetimes year to 100 μsec, they absorb light weakly andare therefore poor luminescent dyes. However, when chelated by anappropriately chosen aromatic donor, energy transfer can occur resultingin high overall luminescent yields. DPA (dipiccolimic acid) is theprototype for such an aromatic Ln chelator, and has excellentphotophysical properties. However, its absorbence maximum is near 260nm, which overlaps the DNA absorption band and is thereforeinappropriate for the proximal CCD approach. Thus, the synthesis ofmodified DPA derivatives with the correct absorption properties andwhich have the capacity to be linked directly or indirectly to thetarget molecules have been developed.

Since three DPA equivalents bind per Ln ion, the preferred approach isto link the modified DPA to a polymeric lattice, which provides forclose spacing of chelators and can be designed to have useful DNA or RNAbinding properties. These results suggest that a fused bicyclic DPAderivative is the candidate of choice.

FIG. 8 is a chemical drawing showing lanthanide chelators. The twoclasses of polymeric latice as illustrated in FIG. 8 can be employed forattachment of DPA derivatives, both based upon the use of syntheticpolypeptides in the 10⁴ MW range. Synthesis can be conducted asdescribed for simple DPA-peptide conjugates. The first polymer is to beused for covalent attachment to RNA via the transamination reaction tocytosine. This peptide lattice can be simple poly-L-lys. The secondapproach involves the coupling of modified DPA to a DNA binding peptide,which can be used to deliver the Ln chelate to RNA by means ofnon-covalent nucleic acid binding. For example, peptides can besynthesized in solution as a Lys₃ Arg₁ random co-polymer (average mw10⁴). Subsequent to the conversion of Lys residues to the modified DPAconjugate, RNA binding can be driven by association with multiple Argequivalents, taking advantage of the known helix selectivity ofpolyarginine. As for ethidium bromide (EB), addition of the non-covalentchelator conjugate can be made after washing to retain hybridizationstringency.

Step 4--Detection System

A detection system relates to parallel detection and/or imaging in thereaction vessel containing the reaction chambers using a proximal largearea detector/imager.

1. General Description

Following the hybridization process of the multiplexed molecularanalysis system, the amount of hybridized target molecules bound to eachbiosite in the reaction chambers of the reaction vessel must bequantitatively determined. The preferred detection/imaging system forquantifying hybridization for the instant invention is proximalcharge-coupled device (CCD) detection/imaging due to the inherentversatility (accommodates chemiluminescence, fluorescent andradioisotope target molecule reporter groups), high throughput, and highsensitivity as further detailed below.

The detection/imaging apparatus used for the multiplexed molecularanalysis system is comprised of a lensless imaging array comprising aplurality of solid state imaging devices, such as an array of CCDs,photoconductor-on-MOS arrays, photoconductor-on-CMOS arrays, chargeinjection devices (CIDs), photoconductor on thin-film transistor arrays,amorphous silicon sensors, photodiode arrays, or the like. The array isdisposed in proximity to the sample (target molecules hybridized to thebiosites) and is comparable in size to the reaction chambers. In thismanner, a relatively large format digital image of the spatialdistribution of the bound target molecules is produced without requiringthe use of one or more lenses between the sample and the imaging array.This apparatus offers:

1) high sensitivity (subattomole DNA detection);

2) high throughput (seconds for complete image acquisition);

3) linear response over a wide dynamic range (3 to 5 orders ofmagnitude);

4) low noise;

5) high quantum efficiency; and

6) fast data acquisition.

Moreover by placing the imaging array in proximity to the sample asillustrated in FIG. 1, the collection efficiency is improved by a factorof at least ten (100 over any lens-based technique such as found inconventional CCD cameras). Thus, the sample (emitter or absorber) is innear contact with the detector (imaging array), thereby eliminatingconventional imaging optics such as lenses and mirrors. This apparatuscan be used for detecting and quantitatively imaging radioisotope,fluorescent, and chemiluminescent labeled molecules, since a lenslessCCD array apparatus is highly sensitive to both photons and x-rayparticles. Hence a single imaging instrument can be used in conjunctionwith numerous molecular labeling techniques, ranging from radioisotopesto fluorescent dyes.

The detection/imaging apparatus invention as taught herein can bedivided into two subclasses. The first subclass entails a staticplatform, whereby a plurality of imaging devices are arranged in arelatively large format area comparable to the sample size.

The second subclass entails a dynamic platform that enables a smallerset of imaging devices to image a relatively large format sample bymoving either the array of imaging devices or sample, relative to oneanother.

Thus, the dynamic embodiment of the detection/imager invention generallyconcerns a method and apparatus for ultrasensitive detection, highresolution quantitative digital imaging and spectroscopy of the spatialand/or temporal distribution of particle emissions or absorption from/bya sample (target molecules) in a relatively large format. The apparatusof this invention includes:

a) a large area detector array for producing a relatively large image ofdetected particle distribution without the use of optical lenses;

b) a scanner for moving either the sensor array or the sample in amanner for efficient imaging; and

c) a source of energy for exciting the sample or providing absorption bythe sample.

Optimally, the ratio of detector array size to sample image is one (1)for a static format and less than one (1) for a dynamic format.

An electronic schematic of the proximal detector/imager to be used withthe multiplexed molecular analysis system is shown in FIG. 9. FIG. 9 isa diagram showing a multiplexed molecular analysis system electronicsschematic. As illustrated in FIG. 9, the reaction vessel is placeddirectly on the fiber optic faceplate which is bonded to the sensorarray. The faceplate provides sensor isolation to accommodate routinecleaning, as well as affording thermal isolation for ultrasensitivedetection under cooled sensor operation. Also the optical faceplate canserve to filter excitation radiation by employing selective coatings.The sensor array is comprised of a plurality of smaller sensors suchthat the composite array approaches the surface area of the reactionvessel. The excitation source serves to excite the fluorescent reportergroups attached to the target molecules. Depending on the chosenreporter groups, the excitation source can be either a UV lamp, laser,or other commonly used light source used by those skilled in the art.The sensor array driver circuitry includes clocking, biasing and gatingthe pixel electrodes within the sensors. The cooling circuitry controlsthe thermoelectric cooler beneath the sensor array to enableultrasensitive detection by providing very low thermal noise. Basically,the user selects the required temperature of operation and throughfeedback circuitry, the sensor array is held constant at suchtemperature. The image receive circuitry is responsible for obtainingthe digital image from the sensor array and includes preamplification,amplification, analog to digital conversion, filtering, multiplexing,sampling and holding, and frame grabbing functions. Finally, the dataprocessor processes the quantitative imaging data to provide therequired parameters for the molecular analysis outcome. Also, a computerdisplay is included for displaying the digital image.

2. Sensor Array Implementations

A preferred embodiment of the detection/imaging sensor array of theinvention consists of a plurality of CCD arrays CCD1 . . . CCDNassembled in a large format module as illustrated in FIGS. 10A-10C. FIG.10A depicts a CCD array with multiple pixels being exposed to a labeledbiological sample 32 which causes the collection of electrons 34 beneaththe respective pixel gate 16. Individual CCD arrays are closely alignedand interconnected in particular geometries to form a relatively large(greater than 1 cm²) format imaging sensors of the linear array type asshown in FIG. 10B or the two dimensional row and column type as shown inFIG. 10C.

Numerous CCD tiling strategies can be explored to determine the besttradeoff analysis between detection throughput and instrument cost. Alarge format tiled array with several wafer scale CCDs would providesimultaneous detection of all biosites within the reaction vessel withinseconds. However the cost of the large (8.5×12.2 cm) CCD sensor arraymay be prohibitive. An engineering compromise is therefore preferred,balancing the use of smaller devices to significantly reduce the cost ofthe tiled array, while also matching the throughput with the otherprocesses in the overall multiplexed molecular analysis system.

As shown in FIG. 10A, each CCD array CCD1 . . . CCDN is formed, in theconventional manner, by growing and patterning various oxide layers 14on a Si wafer/ substrate 12. CCD gate electrodes 10 are then formed bydeposition of polysilicon or other transparent gate material on the gateinsulator or field oxide 14. A dielectric or polymer layer 18,preferably of light transmissive material such as silicon nitride orglass, SiO₂ or polyamide is then formed over the electrodes 16.

Preferably, in a labeled molecule embodiment, a filter shown in dottedlines 17, which may be formed of an aluminum or tungsten metal grating,or dielectric multilayer interference filter, or absorption filter, isformed in the dielectric layer 18 between the surface and the metalelectrode 16. The filter is adapted to block the excitation radiationand pass the secondary emission from the sample 20. In a static platformembodiment, the sensor module remains fixed with respect to the sample.Hence to achieve the relatively large imaging format, a plurality ofimaging devices CCD1 . . . CCDN should be arranged in a module asillustrated in FIGS. 10B and 10C. The module can be packaged for easyinstallation to facilitate multiple modules, each for specificapplications. Various tiling strategies have been documented and can beemployed to minimize the discontinuity between devices, such asdescribed in Burke, et al., "An Abuttable CCD Imager for Visible andX-Ray Focal Plane Arrays," IEEE Trans On Electron Devices, 38(5):1069(May, 1991).

As illustrated in FIG. 10A, a reaction vessel 20 is placed in proximityto the CCD array sensor 10. The sample can be excited by an externalenergy source or can be internally labeled with radioisotopes emittingenergetic particles or radiation, or photons may be emitted by thesample when labeled with fluorescent and chemiluminescent substances.Conversely, direct absorption may be used to determine their presence.In this case, the absence of illuminating radiation on the detector mayconstitute the presence of a particular molecule structure. Preferablythe sample can be physically separated from the CCD detector by thefaceplate which is transparent to the particle emission.

The CCD detection and imaging arrays CCD1 . . . CCDN generateelectron-hole pairs in the silicon 12 (see FIG. 10A) when the chargedparticles or radiation of energy hv shown by the asterisk 32 arisingfrom or transmitted by the sample are incident (arrows 30) on the CCDgates 16. Alternatively, the CCDs can be constructed in a backillumination format whereby the charged particles are incident in thebulk silicon 12 for increased sensitivity. The liberated photoelectrons34 are then collected beneath adjacent CCD gates 16 and sequentiallyread out on a display conventionally.

Silicon based CCDs are preferred as the solid state detection andimaging sensor primarily due to the high sensitivity of the devices overa wide wavelength range of from 1 to 10,000 Å wavelengths. That is,silicon is very responsive to electromagnetic radiation from the visiblespectrum to soft x-rays. Specifically for silicon, only 1.1 eV of energyis required to generate an electron-hole pair in the 3,000 to 11,000 Åwavelength range. Thus for visible light, a single photon incident onthe CCD gate 16 will result in a single electron charge packet beneaththe gate, whereas for soft x-rays, a single beta particle (typically KeVto MeV range) will generate thousands to tens of thousands of electrons.Hence the silicon CCD device provides ultrasensitive detection andimaging for low energy alpha or beta emitting isotopes (³ H, ¹⁴ C, ³⁵ S)as well as high energy alpha or beta emitting isotopes (³² P, ¹²⁵ I).Consequently, the CCD is both a visible imager (applicable tofluorescent and chemiluminescent labeled molecular samples) and aparticle spectrometer (applicable to radioisotope labeled samples aswell as external x-ray radiated samples). Thus, the CCD can providesimultaneous imaging and spectroscopy in the same image.

In addition to the high sensitivity, the CCDs offer a wide dynamic range(up to 5 orders of magnitude) since the charge packet collected beneatheach pixel or gate 16 can range from a few to a million electrons.Furthermore, the detection response is linear over the wide dynamicrange which facilitates the spectroscopy function, since the amount ofcharge collected is directly proportional to the incident photon energy.Hence, no reciprocity breakdown occurs in CCDs, a well-known limitationin photographic film.

3. Scanning Mechanics

To image the reaction vessels with a smaller sized and less expensivesensor array, the reaction vessel can be imaged in a column-by-columnmanner as it is moved across the sensor array with a scanning mechanism.A plurality of imaging devices can be arranged in a module of columns tominimize discontinuity. Also, the scanning can be accomplished withintentional overlapping to provide continuous high resolution imagingacross the entire large format sample area.

EXAMPLE I DIFFERENTIAL DETECTION OF THREE NHS-IMMOBILIZED HAPTENS USINGUNIVERSAL ARRAYS

This example demonstrates reduction to practice of small moleculeuniversal arrays as illustrated in FIGS. 18 and 19. FIG. 18 is agraphical schematic layout of a microarray that will be printed on glassslides using the Hamilton 2200 Microlab robot. This schematic layoutillustrates the relative spatial location/addresses of three separatecovalently immobilized haptens on to the glass substrate (e.g.,digoxigenin, fluorescein, and biotin). The robot will print the array bydepositing 10 nL volumes of each activated hapten (N-hydroxysuccinimideactivated) on to an amino-silanized glass surface thus cerating a 4×4matrix microarray. Each hapten will be deposited by the robot 4 times asillustrated by the schematic. For example, digoxigenin will be depositedat array addresses indicated by address locations A1, B2, C3, and D4.Similarly, fluorescin can be found at address locations A4, B3, C2, andD1 and biotin at B2, B3, B4, and C3 as illustrated in the schematic. Abuffer blank (control) will be deposited at locations A2, A3, D2, andD3. These buffer blanks should not generate a signal on the CCD proximaldetector in the presence of hapten detecting conjugates.

Incubation of these covalently immobilized hapten microarrays with anappropriate bispecific molecule (e.g., hapten recognition site andenzyme reporter site) such as an antibody/enzyme conjugate andsubsequent detection of the appropriate chemiluminescent substrateshould generate an image "pattern" on the CCD detector as predicted bythe schematic addresses shown in FIG. 18. In this example, specificlight generating substrate molecules are localized atpredictable/addressable biosites in the array either individually or ina multiplexed fashion.

Briefly, in order to covalently immobilize the above described haptenmicroarray the following protocol was developed. First, several 22×22 mmsquare glass microscope cover slides (150 μm thick) were washed in acontainer containing ALCONOX detergent solution, and subsequentlytransferred to a clean container containing warm tap water to rinse offthe detergent. This rinse step was followed by two separate briefrinses, first in a container containing 100% acetone, then the slideswere transferred to a rinse in a solution of 100% methanol. The slideswere rinsed one final time in deionized H₂ O to remove traces of organicsolvent. The clean glass slides were then oven dried at 37° C. Afterdrying, the clean slides were then surface derivitized by vacuumdeposition of a solution of 3-aminopropyltrimethoxysilane in a vacuumoven. The slides were laid down in a metal tray on clean lint free papertowels. A 1:3 solution of 3-aminopropyltrimethoxysilane and xylene wasfreshly prepared by mixing 1 ml of 3-aminopropyltrimethoxysilane(Aldrich) with 3 ml of dry p-xylene solvent in a small glass petri dish.The dish was covered with aluminum foil and a small needle puncture wasmade in the foil. This solution was placed in the tray with the glassslides. The tray was subsequently covered with aluminum foil and placedin a NAPCO vacuum oven at 75° C. under 25" of Hg vacuum overnight. Thenext day the amino-silanized glass slides were removed from the vacuumoven and stored in a dry place until used.

In order to robotically dispense and print hapten microarrays, fourseparate activated hapten solutions were made as follows. First,approximately 1 mg of the following compounds were weighed out intoseparate weigh boats: fluorescein-5-(and-6)-carbixamido)hexanoic acid,succinimidyl ester, followed by 1 mg of sulfosuccinimidyl6-(biotinamido) hexanoate and then 1 mg ofdigoxigenin-3-O-methylcarbonyl-γ-aminocaproic acid-N-hydroxysuccinimideester. Each activated hapten was dissolved in 100 μL DMSO. Subsequently50 ul of each hapten was mixed into separate tubes containing 950 ul of0.1 M Na₂ HCO₃ /NaCO₃ buffer at pH 8.05. A blank solution containing 50μL of DMSO into this buffer was also made as a control for dispensing onto the array as described above. Each of the four solutions (100 μL) wasplaced into 16 wells of a microtiter plate. The microtiter plate wasthen placed on the Hamilton 2200 Microlab robot and 10 nL aliquots werecollected and dispensed by the robotic dispensing needle onto theamino-silanized glass cover slides at known address locationsillustrated by the schematic layout in FIG. 18.

Following microfluid dispensing of four separate (identical) glass coverslides by the computer controlled robot needle the arrays were air driedfor 15 minutes. To detect the immobilized haptens the glass slides wererinsed for 10 minutes in 10 ml of 1×TBS+0.1% Tween® 20 (Tris-BufferedSaline, 100 mM Tris-HCl, 150 mM NaCl, pH 7.5). Individual slides werethen incubated with appropriate conjugate dilutions. Image A wasgenerated by incubating one of the slides in 10 ml of a 1:5000 dilutionof streptavidin: horseradish peroxidase conjugate in 1×TBS+0.1% Tween®20. Image B was generated by incubating one of the slides in a 1:5000dilution of anti-digoxigenin: horseradish peroxidase conjugate. Image Cwas generated by incubating in a 1:1000 dilution ofantifluorescin:horseradish peroxidase conjugate. Finally, Image D wasgenerated by incubating a fourth slide simultaneously with all threehorseradish peroxidase conjugates at the above dilutions. Followingconjugate incubation all slides were washed by a 10 minute rinse on aplatform shaker in 10 ml 1×TBS+0.1% Tween® 20 to remove excessconjugates. The slides were then imaged by adding 200 μL of freshly madechemiluminescent substrate (SuperSignal™ Substrate from Pierce Chemical)as recommended by the manufacturer. The slides containing substrate wereimaged by a 10 second integration time at room temperature on theproximal CCD detector described above.

FIG. 19A is a printed computer image showing specific imaging ofbiotin-addressable biosites detected using streptavidin:HRP conjugate(4×4 single well microarray). In FIG. 19A, Image A was generated byincubating the small molecule 4×4 universal array with astreptavidin:HRP conjugate specific for biotin. As seen in this image,only biosites with addresses B1, C1, B4, and C4 known to contain biotin(refer to FIG. 18) are detected using proximal CCD imaging ofchemiluminescent signals. Specific addressing of these biositesgenerates a "box" image pattern.

As shown in FIGS. 19B and 19C, Image B and Image C are two additional4×4 microarrays incubated with the indicated antibody conjugate. FIG.19B is a printed computer image showing specific imaging ofdigoxigenin-addressable biosites detected using anti-digoxigenin:HRPconjugate (4×4 single well microarray). As seen in 19B, only biositeswith addresses A1, B2, C3, and D4 known to contain digoxigenin (refer toFIG. 18) are detected using proximal CCD imaging of chemiluminescentsignals. FIG. 19C is a printed computer image showing specific imagingof fluorescein-addressable biosites detected using anti-fluorescein:HRPconjugate (4×4 single well microarray). As seen in 19C, only biositeswith addresses A4, B3, C2, and D1 known to contain fluorescein (refer toFIG. 18) are detected using proximal CCD imaging of chemiluminescentsignals. Thus, the signals from these two small molecules generate thepredicted "diagonals" as illustrated in FIG. 18.

Additionally, in FIG. 19D, Image D illustrates simultaneous detection ofall three haptens in a single well by simultaneously incubating a single4×4 array with all three conjugates. FIG. 19D is a printed computerimage showing simultaneous imagings of fluorescein, biotin, anddigoxigenin biosites detected using anti-fluorescein, anti-digoxigeninand streptavidin:HRP conjugates (4×4 single well microarray). This imagegenerates the predicted "H" pattern as expected because wells A2, A3, D2and D3 were blank (see FIG. 18).

EXAMPLE II USE--MICROARRAYS IN A MICROPLATE

Several applications of the multiplexed molecular analysis system aredetailed below which can be accommodated with a multiple well microplateserving as the particular reaction vessel. The novelty, however, is theplurality of biosites within each well. That is, each well in themultiple well microtiter plate contains N biosites where N ranges from 2through 1,000. The upper bound is based on the resolution limitationsposed by the bottom substrate of the microtiter plate used inconjunction with the proximal CCD detector/imager.

For example, each well or reaction chamber can contain 96 biosites asshown in FIG. 1l. FIG. 11 is a printed computer image showingmicroarrays within a microplate reaction vessel. One single reactionchamber is shown as an insert. Thus, the reaction vessel essentiallyconsists of microarrays within a microplate which cumulatively affords9,216 (96×96) hybridization experiments per microtiter plate--a 100 to 1multiplexing capacity.

The specific multiplexed microtiter plate reaction vessels to be usedwith proximal imaging are constructed by bonding thin films (typicallyglass or plastics) to conventional bottomless microtiter plates. Allcommercially available microtiter plates tested to date are incompatibleto proximal imaging due to the thickness and composition of the bottomsubstrates.

The biosites are deposited by one of the several methods disclosed,either before or after the bottoms are bonded to the plate. In bothsituations, the probe molecules comprising the individual biosites mustbe attached to the glass or plastic surfaces.

In a preferred embodiment, thin (50-300 μm) vinyl substrates are aminoor epoxy functionalized with silanes similar to glass substrates. Thinvinyl substrates are immersed in a 1-2% aqueous solution of polyvinylalcohol at 65° C. The adsorbed polyvinyl alcohol is then reacted witheither epoxy silane or amino silane, thus functionalizing the polymerichydroxyl groups. Such optically clear vinyl substrates have the distinctadvantage of blocking a large amount of the UV excitation sourceincident on the proximal CCD detector, but allowing the longerwavelengths (e.g. 500-650 nm) to pass through efficiently. This allowsfor greater sensitivity of labeled detector molecules that emit in suchwavelength region.

Nucleic acid probe attachment to glass employs well-known epoxy silanemethods (see FIG. 12) described by Southern and others (U. Maskos elal., Nucleic Acids Res (1992) 20:1679-84; S. C. Case-Green et al,Nucleic Acids Res (1994) 22:131-36; and Z. Guo et al., Nucleic Acids Res(1994) 22:5456-65). FIG. 12 is a diagram showing glass and polypropylenesurface coupling chemistries. With 3' amine-modified probes, covalentsurface densities can be obtained having 10¹¹ molecules/mm² which isnear the theoretical packing density limit. Amino-modified polypropyleneis a convenient alternative to a glass substrate since it is inexpensiveand optically clear above 300 nm. Amine-modified polypropylene can beconverted to a carboxylic acid-modified surface by treatment withconcentrated succinic anhydride in acetonitrile. Amine-modified probe isthen coupled to this surface by standard carbodiimide chemistry in H₂ Oto yield probes at densities near 10⁹ /mm² (see FIG. 12).

EXAMPLE III USE--MULTIPLEXED DIAGNOSTICS

The multiplexed molecular analysis system can be employed forimmunoassay and probe-based diagnostics. For immunoassays, thethroughput of conventional ELISA assays can be increased with themultiplexed microplate format wherein a patient sample can besimultaneously interrogated by numerous antigens/antibodies within asingle reaction chamber (well).

Similarly for probe-based diagnostics, target molecules derived from apatient sample can be dispensed into a single well containing numerousbiosites for diagnosing genetic or infectious diseases. For example,single-stranded nucleic acid probes which are complementary to 96 knownmutations of cystic fibrosis are arranged within a single well in amicroplate. Upon hybridization with the patient's DNA sample, theresulting binding pattern obtained from the proximal CCD detector/imagerindicates the presence of such known mutations.

The system can also be employed for high throughput, probe-basedinfectious disease diagnostics. Here the array of biosites within asingle well in the microtiter plate can comprise DNA probescomplementary to known viral strains. For example, a panel of probes(biosites) is arranged to diagnose a number of sexually transmittablediseases within a single well (reaction chamber). Consequently for asingle microtiter plate, numerous patient samples can be simultaneouslyinterrogated each against a panel of numerous probes to provide a veryrapid, cost effective diagnostic testing platform.

Universal Arrays are perfectly suited for analysis and detection ofmultiple point mutations within a single PCR template. Often technicalconstraints are encountered when attempting to analyze multiple pointmutations from a single PCR amplicon reaction. Most point mutationanalysis techniques such as ribonuclease protection assay, SSCP, orCLEAVASE™ are well suited for detecting a single point mutation peramplicon or DNA template and require lengthy gel-based separationtechniques. The simultaneous, rapid detection of numerous pointmutations within a single PCR amplicon without an expensive, lengthy gelseparation step is well beyond the capability of these technologies.Other newer, non-gel based technologies such as TAQMAN™ are also poorlysuited for multiplexed analysis within a single reaction vessel. FIG. 13illustrates the concept of using Universal Arrays for point mutationanalysis (genotyping) at a single loci. FIG. 13 is a diagram showinggenotyping by universal point mutation scanning. For example purposesonly, FIG. 13 uses a single point mutation biosite to illustrate thistype of analysis but could just as easily be simultaneously carried outon 25 different loci on a single PCR template as illustrated in FIG. 14.

Briefly, as shown in FIG. 13, the PCR template is aliquoted into 4separate tubes (one for each dNTP) containing a standard sequencing mix,with the exception that dideoxynucleotides are not included. Instead, asingle alpha-thio dNTP is substituted in each of the four separatemixes. Each mix also contains a single labeled primer with a universalsequence or "handle" at the 5' end which anneals just one nucleotideaway from the mutation site on the PCR template (note: multiple primerseach with unique universal sequences and complementary to different locion the template is readily accomplished). After standard thermal cycleextension reactions are complete each tube is briefly incubated withsnake venom phosphdiesterase. Only primers and templates that were notextended during the sequencing reaction are vulnerable to digestion bythis 3'-specific exonuclease. Mutation primers containing a 3'thiophosphate ester linkage are highly resistant to digestion.

In this specific example, only the A reaction extended since a T was thenext complementary base on the PCR template. Each digested, sequencingreaction mix in turn is then hybridized to four microtiter wells eachcontaining identical immobilized microarrays complementary to theuniversal primer sequences. In this case, only the microtiter wellhybridized to the A reaction mix gives a positive signal at a biositeloci complementary to the universal handle. In this fashion, up to 96loci could be probed for point mutations on a single PCR template. Bothstrands in the PCR amplicon could be "scanned" in this mannersimultaneously to allow more room for many primers to anneal withoutcompetition for the same hybridization loci on the template. In FIG. 13,"5-DIG" means 5' digoxigenin labeled.

For probe based diagnostics where both multiplexing within a singletarget molecule and low target concentrations are a problem,amplification with either PCR or LCR using the microtiter plate in amicrotiter well concept conjoined to the Universal Array has distinctadvantages. In a preferred embodiment, universal "handles" can besynthesized directly on the 5' end of Polymerase Chain Reaction orLigase Chain Reaction primers and following in situ thermal cycling theamplified products can be simultaneously hybridized to 96 separatebiosites. This format has other diagnostic advantages such ashomogeneous detection of amplified products without having to open orexpose the sample well to the ambient environment.

FIG. 14 is a diagram showing microtiter-based throughput genotyping.Briefly, FIG. 14 illustrates the concept of high throughput genotypingusing microarrays. In practice, 96 separate PCR amplification reactionswould be carried out using genomic DNA templates isolated from 96different patient samples. The figure illustrates the concept ofgenotyping starting with 96 previously robotically purified PCRtemplates from these reactions. Each purified PCR product from each ofthe 96 wells is split into 4 separate aliquots/wells of a 384 wellplate. Each well in this new plate would contain a pre-made sequencingbuffer mixture, 25 individual primers, a thermostable DNA polymerase,and only one of the four αthio-dNTP's. The primers would anneal in ajuxtaposed fashion to the PCR template just one nucleotide away from thenucleotide locus being genotyped. In each of the four wells, thoseprimers juxtaposed next to the included nucleotide in the sequencing mixwould be extended. Following the simultaneous extension of 384reactions, each of the 384 wells is in situ digested with snake venomphosphodiesterase. Only primers in each reaction that had been extendedby a single base are protected from digestion. All other DNA is degradedto mononucleotides. Following a brief thermal denaturation of theexonuclease, the contents of all the wells is robotically transferred toa new 384-well microtiter plate containing sequence complementsmicroarrayed in a 5×5 microarray attached to the bottom of each well.Each of the 25 primers that had not been digested would hybridize to itscorresponding complement in the array and imaged on the CCD detector todefine the genotype at each loci.

FIG. 15 illustrates this homogenous multiplexed approach for thePolymerase Chain Reaction (PCR) simultaneously at 3 different loci. FIG.15 is a diagram showing homogeneous in situ microarray detection ofmultiplexed PCR amplicons. FIG. 15 illustrates specific multiplexhybridization detection of PCR products using microtiter-basedmicroarrays. Briefly, in this figure three separate amplification lociare being detected simultaneously. Each locus (e.g, PCR LOCUS 1) isdefined by two specially modified amplification primers that define theends of the amplified PCR product. One primer in the pair, contains afluorescently detectable label such as fluorescein. The other primer inthe pair contains two domains, one is a unique universal sequencecomplementary to a capture probe arrayed at the bottom of a singlemicrotiter well and the other domain specific for templateamplification. The universal sequence is attached to the amplificationprimer in a 5' to 5' linkage so that when the polymerase is amplifyingthe region of interest it does not jump over this specialized juncture,leaving the universal sequence as a single stranded motif. If aparticular template in a sample well being amplified contains bothprimer loci (i.e., detection and capture sites), then a PCR product willbe generated that can simultaneously hybridize and be detected to acomplementary member of a universal capture array by the CCD proximaldetector. Since only PCR amplicons hybridized to members of theuniversal array at the bottom of each well are proximal to the detector,the assay requires no special separation step to detect hybridizedamplicons and thus becomes homogenous in nature.

Similarly, FIG. 16 illustrates this multiplexed concept with Gap-LigaseChain Reaction (G-LCR). FIG. 16 is a diagram showing homogeneous in situmicroarray detection of multiplexed gap-ligase chain reaction products.The ability to detect hybridization events homogeneously is provided bythe fact that only molecules proximally associated with specificbiosites can be imaged by the detector. FIG. 16 illustrates specificmultiplex hybridization detection of Gap-Ligase Chain Reaction productsusing microtiter-based microassays. Similarly, as described previouslyfor PCR products (see FIG. 15), this figure illustrates the assay atthree separate ligation-dependent amplification loci simultaneously.Each locus (e.g., LOCUS 1) is defined by two specially modified primersthat define the ends of the gap ligase chain reaction product. Oneprimer in the pair, contains a fluorescently detectable label such asfluorescein. The other primer in the pair contains two domains, one is aunique universal sequence complementary to a capture probe arrayed atthe bottom of a single microtiter well and the other domain is specificfor a region on the template being detected. The universal sequenceattached to this primer serves as a sequence specific single strandedhandle. When the template is present in the sample then sequencedirected ligation will join both the label and the universal handle intoa single product. After many cycles this amplified ligated product canbe simultaneously hybridized and detected to its complementary member ona universal capture array immobilized to the bottom of a microtiter welland imaged by the CCD proximal detector. Since only ligated productshybridized to members of the universal array at the bottom of each wellare proximal to the detector, the assay requires no special separationstep to detect hybridized amplicons and thus becomes homogenous innature.

EXAMPLE IV DRUG DISCOVERY/SCREENING ANALYSIS

In this example, a small molecule Universal Array could use highaffinity, commercially available antibodies to numerous haptens,steroids, or small molecule drugs. A partial list of 48 representativecompounds are enumerated in Table 1 for which specific antibodies areavailable. This table is only a partial list of commercially availableantibodies to haptens, steroid hormones and other small molecule drugs.

                  TABLE 1                                                         ______________________________________                                        fluorescein                                                                              dinitrophenol                                                                            amphetamine                                                                              barbiturate                                    acetaminophen acetohexamide desipramine lidocaine                             digitoxin chloroquinine quinine ritalin                                       phenobaribital phenytoin fentanyl phencyclidine                               methamphetamine metaniphrine digoxin penicillin                               tetrahydrocannibinol tobramycin nitrazepam morphine                           Texas Red TRITC primaquine progesterone                                       bendazac carbamazepine estradiol theophylline                                 methadone methotrexate aldosterone norethisterone                             salicylate warfarin cortisol testosterone                                     nortrptyline propanolol estrone androstenedione                               digoxigenin biotin thyroxine triiodothyronine                               ______________________________________                                    

Small molecule Universal Arrays are made by covalent attachment of smallmolecules such as those found in Table 1 to substrate surfaces.Immobilization of haptens, steroids, or drugs is accomplished byintroducing a functionalized moiety at one end of the small molecule.These moieties are well known to those skilled in the art (e.g.N-hydroxy-succinimide, maleimide, isothiocyanate, iodoacetamide or otheramine or sulfur reactive moieties). Small functionalized molecules ordrugs can then be reacted with NH₂ or SH₂ derivitized plastic or glasssubstrates. Some specific examples of such commercially availableactivated haptens include NHS-fluorescein, NHS-biotin, NHS-digoxigenin,maleimide-biotin, and maleimide-tetramethylrhodamine.

Following deposition of the individual small molecule biosites, abispecific ligand can be used to spatially localize specific bindingevents to given biosites. The bispecific ligand can comprise, but is notlimited to, antibody-antibody conjugates, antibody-receptor,antibody-streptavidin, antibody-peptide, antibody-small moleculeconjugates or bispecific antibodies.

The bispecific ligand is specific to both the immobilized hapten or drugon the substrate surface (biosite) and the analyte being screened.Examples of Universal Array screening are diagramed in FIG. 17. FIG. 17is a diagram showing small molecule universal array (drugscreening/discovery). FIG. 17 illustrates the basic small moleculeUniversal Array concept using four different immobilized haptens in asingle well. Various bispecific molecules are diagramed for illustrationpurposes. FIG. 17 illustrates four separate and distinct haptensimmobilized at the bottom of each of 96 wells of a microtiter plate.Each locus or biosite in the array is defined by four unique immobilizedhaptens illustrated in this example by fluorescein, digoxigenin, 2,4dinitrophenol, and TRITC. Bispecific molecules uniquely specific forboth the immobilized hapten and another labeled analyte in the sampleare added to each well. In this fashion, different multiple analytes canbe simultaneously detected and their presence indicated by signals atspecific hapten biosites. In this example, 96 individual samples can beassayed for four different analytes simultaneously. As shown, thefluorescein biosite detects a labeled receptor (protein) analyte, boththe 2,4 dinitrophenol and digoxigenin haptens allow for the simultaneousdetection or presence of two additional types of protein receptors inthe sample. Finally, the TRITC hapten allows for detection and presenceof a specific enzyme substrate via an intervening enzyme conjugate. Onceagain, the proximal mode of detection allows for homogenous imaging ofonly those binding events at the surface of the array. The advantages ofsuch a multiplexed immunological approach is the exquisite specificityand variety of small molecules that comprise such a Universal Arrayusing non-DNA based recognition of biosites.

Actual reduction to practice of small molecule Universal Arrays isillustrated in FIGS. 18 and 19 and described in Example I above.

EXAMPLE V USE--GENE EXPRESSION ANALYSIS

The multiplexed molecular analysis system is also useful for analyzingthe expression of hundreds of different mRNA species in a single tissuesample within a single well of a microtiter plate. Here syntheticnucleic acids form the distinct biosites which constitute numeroushighly sensitive and selective hybridization analyses per sample,employing only 50 μL of sample extract. Such massive hybridizationanalyses enables the discovery and employment of numerous biomarkers forspecific diseases such as cancer. Essentially, the search for biomarkersof early phase lung cancer becomes an iterative, combinatorial process.For lung cancer and other epithelial disease, several hundred mRNAs areanalyzed for their value as biomarkers at relatively low cost. In suchan iterative process, the biostatistician becomes the end-user of thetechnology and a central component in the development of the final setof mRNA biomarkers. Once an mRNA biomarker set is discovered by thisiterative approach, the technology is naturally suited for low cost,high throughput screening of large patient populations with the mRNAbiomarker set of choice.

EXAMPLE VI USE--CELL SORTING

Conversely, intact cells are analyzed utilizing the multiplexed formatof this invention. Specifically, most "cell enrichment" protocolsinvolve either double label flow cytometry, or physical separation ofcells via affinity chromatography of some kind. Both require access toan antibody which is specific to the cell type of interest.

In a multiplexed microplate format, the cell-specific antibodies arearranged in a matrix fashion within the reaction chamber (single well inthe 96 well microplate). The key to making the cellular analysis work iscreating a situation wherein such antibody arrays retain the capacityfor high affinity and high selectivity binding of intact cells.

The procedure is to add a complex cellular mixture, e.g., a biologicalsample (for example, blood or sputum), to such an antibody matrix, thenwith some local mixing, allowing the cells to bind to the surface. Ifcells bind to such a matrix with good affinity and selectivity, they arethen fixed to the matrix, permeabilized, and subjected to labeled probehybridization (or PCR) in a fashion which is nearly identical to themethods which are currently used to analyze DNA or RNA in cells formicroscopy or flow cytometry.

The principle benefit of the multiplexed format is that many differentcell types arc separated in a single well of a microtiter plate.

EXAMPLE VII USE--MICROORGANIC MONITORING

Microorganism monitoring applications can also be addressed by themultiplexed molecular analysis system. In particular for monitoring air,water, and food for microorganisms, the system can rapidly and costeffectively provide detection and quantification of complex biologicalmixtures. An example would be a ribosomal RNA probe-based assay in whichnucleic acid probes serving as the biosites are chosen to selectivelycapture RNA of characteristic microorganisms.

Basically, the procedure is initiated by preparing the microbial rRNAsample for hybridization to the biosite array within the reactionchamber. Following specific binding of the fluorescently labeledmicrobial RNA to the probe array, a two dimensional image results thatuniquely characterizes the sample. The analyzer output is the microbialspectrum, consisting of the amount and type of microorganisms present inthe sample.

The rationale for the proposed approach to simultaneous monitoring ofmicroorganisms includes:

1) Fast microbial analysis can be achieved due to the avoidance ofstandard cell cultivation procedures which require days to perform.Moreover, the proposed highly sensitive proximal CCD detectionprocedure, combined with the inherent amplification property of rRNA,reduces the combined sample preparation, assay, and detection time fromdays to hours.

2) Simultaneous microbial monitoring can be achieved due to the highdensity arrays that support hundreds of immobilized probes per cm² tofacilitate multiple microorganism detection and identification in a highthroughput manner.

3) Minimal labor and training is required since no cell culturing orgel-based sequencing is required. Instead, an operator merely subjectsthe prepared sample to automated hybridization, washing, and dryingprocesses to obtain the microbial spectrum.

4) Minimal equipment is necessary since the probe-based assay isintegrated with the proximal CCD detection device, thereby alleviatingtraditional macro-detection techniques such as epifluorescent andconfocal microscopy.

The following references may facilitate the understanding or practice ofthe certain aspects and/or embodiments of this invention. Inclusion of areference in this list is not intended to and does not constitute anadmission that the reference represents prior art with respect to thepresent invention.

Hansell, U.S. Pat. No. 2,512,743

D. Bogg, F. Talke, IBM Jour. Res. Develop. (1984) 29:214-321

Burke, el al., "An Abuttable CCD Imager for Visible and X-Ray FocalPlane Arrays," IEEE Trans. On Electron Devices, 38(5):1069 (May, 1991).

Maskos, U., et al., Nucleic Acids Res. 20:1679-1684 (1992).

Stephen C. Case-Green, et al., Nucleic Acids Res. 22:131-136 (1994).

Guo, Z., et al., Nucleic Acids Res. 22:5456-5465 (1994).

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A device for preparing a reaction substratehaving biosites for conducting multiplexed microassays to determinebinding between a target analyte and a capture probe, including:aplurality of capillary tubes, each tube having a proximal end and adistal end; an attachment site for holding said capillary tubes at apoint spaced from the distal ends of such capillary tubes; an arraytemplate for slidably holding each capillary tube near its distal end,and for allowing the distal end of each capillary tube to move withrespect to the attachment site; at least one manifold for positioningthe proximal end of each capillary tube within a corresponding supplychamber, wherein each supply chamber is capable of supplying a liquidreagent to at least one corresponding capillary tube; and a positioningdevice for precisely positioning the array template and said capillarytubes with respect to said reaction substrate and depositing liquidreagents from said distal end of said capillary tubes onto said reactionsubstrate as biosites.
 2. The device of claim 1, wherein said pluralityof capillary tubes comprises about 2 to about 10,000 tubes, having acenter to center spacing of about 80 μm to about 5 mm.
 3. The device ofclaim 1, wherein each supply chamber supplies only one capillary tube.4. The device of claim 1, wherein a supply chamber supplies a pluralityof capillary tubes.
 5. The device of claim 1, wherein said capillarytubes comprise stainless steel, plastic, rubber, glass, or fused silicacoated with polyimide.
 6. The device of claim 1, wherein said deviceincludes a plurality of array templates.
 7. The device of claim 1,further comprising a positioning device for precisely positioning saidreaction substrate.
 8. The device of claim 1, wherein said capillarieshave an inside diameter of about 10 to about 200 μm, and an outsidediameter of about 80 to about 500 μm.
 9. The device of claim 1, whereinsaid array template comprises an array of sleeves, each sleeve having aninside diameter sufficient to permit a capillary to slide through, and alength sufficient to allow a precise pattern to be maintained whiledepositing fluids onto said reaction substrate.
 10. The device of claim1, wherein said array template comprises a plurality of holes formed ina rigid material.
 11. The device of claim 1, wherein said array templatecomprises a rigidly formed or held mesh.
 12. The device of claim 1,further comprising a housing for containing said supply chambers. 13.The device of claim 12, wherein said housing is capable of maintainingan inert atmosphere.
 14. The device of claim 12, wherein said housing iscapable of maintaining an elevated or reduced temperature.
 15. Thedevice of claim 12, wherein said housing may be pressurized to apredetermined pressure.
 16. The device of claim 15, wherein saidpressure is modulated to control the flow of liquid reagents throughsaid capillary tubes.
 17. The device of claim 1, wherein said supplychambers are positioned higher than the distal end of said capillarytubes to provide a pressure head.
 18. The device of claim 1, whereindeposit of liquid reagent is controlled by a means for electrophoresis.19. The device of claim 1, wherein deposit of liquid reagent iscontrolled by a means for electro-osmosis.
 20. The device of claim 1,wherein said device includes multiple sets of supply chambers, each forsupplying liquid reagent to a subset of said capillary tubes.
 21. Thedevice of claim 1, wherein said capillary tubes are free to flex betweensaid attachment site and said array template.
 22. The device of claim 1,wherein the reaction substrate is pre-etched to define a pattern ofreactive areas matching the geometry of the deposited biosites.
 23. Thedevice of claim 1, wherein said biosites are deposited substantiallysimultaneously.
 24. The device of claim 1, wherein each biosite isfluidically isolated from each other biosite.
 25. The device of claim 1,wherein the liquid reagent comprises at least one capture probe.
 26. Anapparatus for preparing a reaction substrate for use as an assay device,including:an attachment site for holding a plurality of flexiblecapillary tubes at a point spaced from the distal ends of such capillarytubes; a structure for positioning the proximal end of each capillarytube within a corresponding supply chamber, each supply chamber capableof supplying a liquid reagent to at least one corresponding capillarytube; a print head for holding said plurality of capillary tubes neartheir distal ends and for precisely positioning the distal ends of saidcapillary tubes with respect to said reaction substrate to depositliquid reagents onto said reaction substrate as biosites.
 27. Theapparatus of claim 26, where the print head includes an ink-jetdeposition device.